Beta-glucosidase and uses thereof

ABSTRACT

The application relates to a polypeptide having beta-glucosidase activity, its method of production and its uses.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application is a Continuation of U.S. application Ser. No.15/781,857, filed 6 Jun. 2020, which is a National Stage entry ofInternational Application No. PCT/EP2016/080240, filed 8 Dec. 2016,which claims priority to European Patent Application No. 15198788.0,filed 10 Dec. 2015, European Patent Application No. 15198826.8, filed 10Dec. 2015, European Patent Application No. 15198902.7, filed 10 Dec.2015, and European Patent Application No. 15198893.8, filed 10 Dec.2015. The disclosure of the priority applications are incorporated intheir entirety herein by reference.

REFERENCE TO SEQUENCE LISTING SUBMITTED AS A COMPLIANT ASCII TEXT FILE(.txt)

Pursuant to the EFS-Web legal framework and 37 CFR §§ 1.821-825 (seeMPEP § 2442.03(a)), a Sequence Listing in the form of an ASCII-complianttext file (entitled “Sequence_Listing_2919208-475001_ST25.txt” createdon 22 Jul. 2020, and 40,288 bytes in size) is submitted concurrentlywith the instant application, and the entire contents of the SequenceListing are incorporated herein by reference.

FIELD

The application relates to polypeptides having beta-glucosidase activityand polynucleotides encoding the polypeptides. Also included in theapplication are nucleic acid contructs, vectors and host cellscomprising the polynucleotides. The application also relates to methodsof producing the polypeptides as well as methods of using thepolypeptides.

BACKGROUND

Carbohydrates constitute the most abundant organic compounds on earth.However, much of this carbohydrate is sequestered in complex polymersincluding starch and a collection of carbohydrates and lignin known aslignocellulose. The main carbohydrate components of lignocellulose arecellulose, hemicellulose and pectins. These complex polymers are oftenreferred to collectively as lignocellulose.

Bioconversion of lignocellulosic biomass to a sugar that is subsequentlyfermented to produce alcohol as an alternative to liquid fuels hasattracted an intensive attention of researchers since 1970s, when theoil crisis broke out because of decreasing output of petroleum by theOPEC. Ethanol has been widely used as a 10% blend to gasoline in the USAor as a neat fuel for vehicles in Brazil in the last two decades. Morerecently, the use of E85, an 85% ethanol blend has been implementedespecially for clean city applications.

The importance of biofuel will increase in parallel with increases inprices for oil and the gradual depletion of its sources. Additionally,fermentable sugars are being used to produce plastics, polymers andother bio-based products and this industry is expected to growsubstantially therefore increasing the demand for abundant low costfermentable sugars which can be used as a feedstock in lieu ofpetroleum-based feedstocks.

The sequestration of large amounts of carbohydrates provides a plentifulsource of potential energy in the form of sugars, both five carbon andsix carbon sugars, which could be utilized for numerous industrialmethods. However, the enormous energy potential of these carbohydratesis currently under-utilized, because the sugars are locked in complexpolymers and hence are not readily accessible for fermentation.

Regardless of the type of cellulosic feedstock, the cost and hydrolyticefficiency of enzymes are major factors that restrict thecommercialization of biomass bioconversion methods. The production costsof microbially produced enzymes are tightly connected with aproductivity of the enzyme-producing strain, the specific activity ofthe enzymes, the mode of action of the enzyme and the final activityyield in the fermentation broth.

In spite of the continued research of the last few decades to understandenzymatic lignocellulosic biomass degradation and cellulase production,it remains desirable to develop new highly active cellulases.

The present application fulfils this need in that it provides newpolypeptides comprising beta-glucosidase activity and polynucleotidesencoding the polypeptides.

SUMMARY

The present application relates to a variant polypeptide comprising asubstitution at one or more positions corresponding to positions 90,103, 142, 335, 485 and 606 of the polypeptide of SEQ ID NO: 2. In anembodiment the variant polypeptide has beta-glucosidase activity.

The present application relates to a variant polypeptide as describedherein, which is a variant of a parent polypeptide which hasbeta-glucosidase activity and which comprises at least 60%, at least65%, at least 70%, at least 71%, at least 72%, at least 73%, at least74%, at least 75%, at least 76%, at least 77%, at least 78%, at least79%, at least 80%, at least 81%, at least 82%, at least 83%, at least84%, at least 85%, at least 86%, at least 87%, at least 88%, at least89%, at least 90%, at least 91%, at least 92%, at least 93%, at least94%, at least 95%, at least 96%, at least 97%, at least 98% or at least99% sequence identity to the polypeptide of SEQ ID NO: 2.

The present application relates to a variant polypeptide as describedherein, which comprises at least 60%, at least 65%, at least 70%, atleast 71%, at least 72%, at least 73%, at least 74%, at least 75%, atleast 76%, at least 77%, at least 78%, at least 79%, at least 80%, atleast 81%, at least 82%, at least 83%, at least 84%, at least 85%, atleast 86%, at least 87%, at least 88%, at least 89%, at least 90%, atleast 91%, at least 92%, at least 93%, at least 94%, at least 95%, atleast 96%, at least 97%, at least 98% or at least 99% sequence identityto the polypeptide of SEQ ID NO: 2.

The present application relates to a variant polypeptide as describedherein, wherein the position corresponding to position 90 of thepolypeptide of SEQ ID NO: 2 is substituted to L. The present applicationrelates to a variant polypeptide as described herein, wherein theposition corresponding to position 103 of the polypeptide of SEQ ID NO:2 is substituted to A. The present application relates to a variantpolypeptide as described herein, wherein the position corresponding toposition 142 of the polypeptide of SEQ ID NO: 2 is substituted to S. Thepresent application relates to a variant polypeptide as describedherein, wherein the position corresponding to position 335 of thepolypeptide of SEQ ID NO: 2 is substituted to V. The present applicationrelates to a variant polypeptide as described herein, wherein theposition corresponding to position 485 of the polypeptide of SEQ ID NO:2 is substituted to I. The present application relates to a variantpolypeptide as described herein, wherein the position corresponding toposition 606 of the polypeptide of SEQ ID NO: 2 is substituted to A. Thepresent application also relates to a variant polypeptide as describedherein, which comprises one or more of these substitutions.

The present application relates to a variant polypeptide as describedherein, which comprises substitution M90L. The present applicationrelates to a variant polypeptide as described herein, which comprisessubstitution N103A. The present application relates to a variantpolypeptide as described herein, which comprises substitution G142S. Thepresent application relates to a variant polypeptide as describedherein, which comprises substitution M335V. The present applicationrelates to a variant polypeptide as described herein, which comprisessubstitution M485I. The present application relates to a variantpolypeptide as described herein, which comprises substitution L606A.

The present application also relates to a polynucleotide which encodes avariant polypeptide as described herein.

The present application also relates to a nucleic acid construct orvector comprising a polynucleotide as described herein.

The present application also relates to a host cell comprising apolynucleotide as described herein or a nucleic acid construct or avector as described herein. The host cell may be a fungal cell.

The present application also relates to a method of producing a variantpolypeptide as described herein, which method comprises the steps of (a)cultivating a host cell as described herein under conditions conduciveto the production of the variant polypeptide, and (b) optionally,recovering the variant polypeptide.

The present application also relates to a composition comprising (i) avariant polypeptide as described herein, and (ii) a cellulase and/or ahemicellulase and/or a pectinase. The cellulase may be selected from thegroup consisting of a lytic polysaccharide monooxygenase, acellobiohydrolase I, a cellobiohydrolase II, an endo-beta-1,4-glucanase,a beta-glucosidase and a beta-(1,3)(1,4)-glucanase or any combinationthereof and the hemicellulase may be selected from the group consistingof an endoxylanase, a beta-xylosidase, an alpha-L-arabinofuranosidase,an alpha-D-glucuronidase, an acetyl-xylan esterase, a feruloyl esterase,a coumaroyl esterase, an alpha-galactosidase, a beta-galactosidase, abeta-mannanase, a beta-mannosidase or any combination thereof. Thecomposition may be a whole fermentation broth.

The present application also relates to a method for the treatment of asubstrate comprising cellulose and/or hemicellulose which methodcomprises the step of contacting the substrate with a variantpolypeptide as described herein and/or a composition as describedherein.

The present application also relates to a method of producing afermentation product, which method comprises the steps of (a) treating asubstrate using the method for the treatment of a substrate comprisingcellulose and/or hemicellulose as described herein, and (b) fermentingthe resulting material to produce the fermentation product.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1: Map of pGBTOP for expression of genes in A. niger. Depicted arethe gene of interest (GOI) expressed from the glucoamylase promoter(PglaA). In addition, the glucoamylase flank (3′-glaA) of the expressioncassette is depicted. In this application a gene of interest is thecoding sequence of a polypeptide as described herein.

DETAILED DESCRIPTION

Throughout the present specification and the accompanying claims, thewords “comprise” and “include” and variations such as “comprises”,“comprising”, “includes” and “including” are to be interpretedinclusively. That is, these words are intended to convey the possibleinclusion of other elements or integers not specifically recited, wherethe context allows.

The articles “a” and “an” are used herein to refer to one or to morethan one (i.e. to one or at least one) of the grammatical object of thearticle. By way of example, “an element” may mean one element or morethan one element.

The term “derived from” also includes the terms “originated from”,“obtained from”, “obtainable from”, “isolated from”, and “created from”,generally indicates that one specified material find its origin inanother specified material or has features that can be described withreference to another specified material. As used herein, a substance(e.g., a polynucleotide or polypeptide) “derived from” a microorganismpreferably means that the substance is native to that microorganism.

The present application relates to a variant polypeptide comprising asubstitution at one or more positions corresponding to positions 90,103, 142, 335, 485 and 606 of the polypeptide of SEQ ID NO: 2. In anembodiment the variant polypeptide has beta-glucosidase activity. Thepresent application relates to a variant polypeptide comprising asubstitution at one or more positions corresponding to positions90+335+485, 103, 142, and 606 of the polypeptide of SEQ ID NO: 2. In anembodiment the variant polypeptide has beta-glucosidase activity.

The present application relates to a variant polypeptide as describedherein, which is a variant of a parent polypeptide which hasbeta-glucosidase activity and which comprises at least 60%, at least65%, at least 70%, at least 71%, at least 72%, at least 73%, at least74%, at least 75%, at least 76%, at least 77%, at least 78%, at least79%, at least 80%, at least 81%, at least 82%, at least 83%, at least84%, at least 85%, at least 86%, at least 87%, at least 88%, at least89%, at least 90%, at least 91%, at least 92%, at least 93%, at least94%, at least 95%, at least 96%, at least 97%, at least 98% or at least99% sequence identity to the polypeptide of SEQ ID NO: 2.

The present application relates to a variant polypeptide as describedherein, which comprises at least 60%, at least 65%, at least 70%, atleast 71%, at least 72%, at least 73%, at least 74%, at least 75%, atleast 76%, at least 77%, at least 78%, at least 79%, at least 80%, atleast 81%, at least 82%, at least 83%, at least 84%, at least 85%, atleast 86%, at least 87%, at least 88%, at least 89%, at least 90%, atleast 91%, at least 92%, at least 93%, at least 94%, at least 95%, atleast 96%, at least 97%, at least 98% or at least 99% sequence identityto the polypeptide of SEQ ID NO: 2. In an embodiment the variantpolypeptide as described herein comprises less than 100% sequenceidentity to to the polypeptide of SEQ ID NO: 2.

The present application relates to a variant polypeptide as describedherein, wherein the position corresponding to position 90 of thepolypeptide of SEQ ID NO: 2 is substituted to L. The present applicationrelates to a variant polypeptide as described herein, wherein theposition corresponding to position 103 of the polypeptide of SEQ ID NO:2 is substituted to A. The present application relates to a variantpolypeptide as described herein, wherein the position corresponding toposition 142 of the polypeptide of SEQ ID NO: 2 is substituted to S. Thepresent application relates to a variant polypeptide as describedherein, wherein the position corresponding to position 335 of thepolypeptide of SEQ ID NO: 2 is substituted to V. The present applicationrelates to a variant polypeptide as described herein, wherein theposition corresponding to position 485 of the polypeptide of SEQ ID NO:2 is substituted to I. The present application relates to a variantpolypeptide as described herein, wherein the position corresponding toposition 606 of the polypeptide of SEQ ID NO: 2 is substituted to A. Thepresent application also relates to a variant polypeptide as describedherein, which comprises one or more of these substitutions. For example,the present application relates to a variant polypeptide as describedherein, wherein the position corresponding to position 90 of thepolypeptide of SEQ ID NO: 2 is substituted to L, the positioncorresponding to position 335 of the polypeptide of SEQ ID NO: 2 issubstituted to V and the position corresponding to position 485 of thepolypeptide of SEQ ID NO: 2 is substituted to I.

The present application relates to a variant polypeptide as describedherein, which comprises substitution M90L. The present applicationrelates to a variant polypeptide as described herein, which comprisessubstitution N103A. The present application relates to a variantpolypeptide as described herein, which comprises substitution G142S. Thepresent application relates to a variant polypeptide as describedherein, which comprises substitution M335V. The present applicationrelates to a variant polypeptide as described herein, which comprisessubstitution M485I. The present application relates to a variantpolypeptide as described herein, which comprises substitution L606A. Thepresent application also relates to a variant polypeptide as describedherein, which comprises one or more of these substitutions. For example,the present application relates to a variant polypeptide as describedherein, which comprises substitution M90L, M335V and M485I.

The present application relates to a variant polypeptide as describedherein, which additionally differs in 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11,12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29,30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47,48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65amino acids from the amino acid sequence of its parent polypeptide. Inan embodiment the parent polypeptide has beta-glucosidase activity andcomprises at least 60%, at least 65%, at least 70%, at least 71%, atleast 72%, at least 73%, at least 74%, at least 75%, at least 76%, atleast 77%, at least 78%, at least 79%, at least 80%, at least 81%, atleast 82%, at least 83%, at least 84%, at least 85%, at least 86%, atleast 87%, at least 88%, at least 89%, at least 90%, at least 91%, atleast 92%, at least 93%, at least 94%, at least 95%, at least 96%, atleast 97%, at least 98% or at least 99% sequence identity to thepolypeptide of SEQ ID NO: 2.

In an embodiment the parent polypeptide is a fugal polypeptide. In anembodiment the parent polypeptide is a fungal beta-glucosidase. In anembodiment the parent polypeptide is a GH3 beta-glucosidase. In anembodiment the parent polypeptide is a Rasamsonia beta-glucosidase. Inan embodiment the parent polypeptide is a Rasamsonia emersoniibeta-glucosidase. In an embodiment the parent polypeptide comprises theamino acid sequence of SEQ ID NO: 2. In an embodiment the parentpolypeptide consists of the amino acid sequence of SEQ ID NO: 2. Theamino acid sequence of SEQ ID NO: 2 discloses the amino acid sequence ofa wild-type (i.e. unmutated) Rasamsonia emersonii beta-glucosidase.Rasamsonia emersonii beta-glucosidase can also be called Talaromycesemersonii beta-glucosidase. The amino acid sequence of SEQ ID NO: 2 isidentical to the amino acid sequence of SEQ ID NO: 5 as disclosed in WO2011/098577. The variant polypeptide as described herein may be avariant of this beta-glucosidase.

Furthermore, the present application provides a polynucleotide encodinga variant polypeptide as described herein. The nucleotide sequence ofSEQ ID NO: 1 encodes the amino acid sequence of SEQ ID NO: 2. Thenucleotide sequence of SEQ ID NO: 1 is identical to the nucleotidesequence of SEQ ID NO: 6 as disclosed in WO 2011/098577. The nucleotidesequence of SEQ ID NO: 6 as disclosed in WO 2011/098577 encodes theamino acid sequence of SEQ ID NO: 5 as disclosed in WO 2011/098577.

The application also provides a nucleic acid construct or vectorcomprising a polynucleotide as described herein.

A host cell comprising the polynucleotide as described herein or thenucleic acid construct or vector as described herein is also part of thepresent application. In an embodiment the host cell as described hereinis a fungal cell, preferably a fungal cell selected from the groupconsisting of the genera Acremonium, Agaricus, Aspergillus,Aureobasidium, Chrysosporium, Coprinus, Cryptococcus, Filibasidium,Fusarium, Humicola, Magnaporthe, Mucor, Myceliophthora, Neocaffimastix,Neurospora, Paecilomyces, Penicillium, Piromyces, Panerochaete,Pleurotus, Schizophyllum, Talaromyces, Rasamsonia, Saccharomyces,Thermoascus, Thielavia, Tolypocladium, and Trichoderma. In a host cellas described herein one or more genes can be deleted, knocked-out ordisrupted in full or in part.

The application also provides a method of producing a variantpolypeptide as described herein, which method comprises cultivating ahost cell as described herein under conditions which allow forexpression of the variant polypeptide and, optionally, recovering theexpressed variant polypeptide. The application also relates to a methodof producing a variant polypeptide as described herein, which methodcomprises the steps of (a) cultivating a host cell as described hereinunder conditions conducive to the production of the variant polypeptide,and (b) optionally, recovering the variant polypeptide.

Furthermore, the application provides a composition comprising (i) avariant polypeptide as described herein, and (ii) a cellulase and/or ahemicellulase and/or a pectinase. In an embodiment the cellulase isselected from the group consisting of a lytic polysaccharidemonooxygenase, a cellobiohydrolase I, a cellobiohydrolase II, anendo-beta-1,4-glucanase, a beta-glucosidase and abeta-(1,3)(1,4)-glucanase or any combination thereof and thehemicellulase is selected from the group consisting of an endoxylanase,a beta-xylosidase, an alpha-L-arabinofuranosidase, analpha-D-glucuronidase, a feruloyl esterase, a coumaroyl esterase, analpha-galactosidase, a beta-galactosidase, a beta-mannanase, abeta-mannosidase or any combination thereof.

The variant polypeptides as described herein may be used in industrialmethods as described in more detail herein.

Additionally, the application provides a method for the treatment of asubstrate comprising cellulose and/or hemicellulose which methodcomprises the step of contacting the substrate with a variantpolypeptide as described herein and/or a composition as describedherein. In an embodiment the substrate is a cellulosic material. Inanother embodiment the substrate is a lignocellulosic material. In anyevent, the treatment results in the production of sugar.

Another aspect of the application relates to the use of a variantpolypeptide as described herein and/or a composition as described hereinto produce sugar from a lignocellulosic material.

The application also provides a method for producing sugar fromcellulosic material which method comprises contacting the cellulosicmaterial with a variant polypeptide as described herein and/or acomposition as described herein and producing sugar from the cellulosicmaterial.

The application also provides a method for producing sugar fromlignocellulosic material which method comprises contacting thelignocellulosic material with a variant polypeptide as described hereinand/or a composition as described herein and producing sugar from thelignocellulosic material.

The sugar produced may be used in a fermentation method as describedherein. Accordingly, the application provides a method for producing afermentation product, which method comprises the steps of treating asubstrate using a method as described above, and fermenting theresulting material to produce a fermentation product. The resultingmaterial may comprise sugar.

A polypeptide as described herein or a composition as described hereinmay also be used, for example, in the preparation of a food product, inthe preparation of a detergent, in the preparation of an animal feed, inthe treatment of pulp, in the manufacture of paper or in the preparationof a fabric or textile or in the cleaning thereof.

The application also provides a material obtainable by contacting aplant material or lignocellulosic material with a variant polypeptide asdescribed herein and/or a composition as described herein; a food orfeed comprising a variant polypeptide as described herein and/or acomposition as described herein; and a plant or a part thereof whichcomprises a polynucleotide, a variant polypeptide, a nucleic acidconstruct, a vector or a host cell as described herein.

In case a variant polypeptide as described herein comprises asubstitution at one or more positions corresponding to positions 90,103, 142, 335, 485 and 606 of the polypeptide of SEQ ID NO: 2, thismeans that the variant polypeptide comprises a substitution at one ofthese positions, but also includes variant polypeptides that comprise asubstitution at more than one of these positions. For example, a variantpolypeptide as described herein may comprise a substitution at aposition corresponding to positions 90, 142, and 485 of the polypeptideof SEQ ID NO: 2. Any combination of the listed positions is possible.Such combinations may have a synergistic effect.

As described above, when a variant polypeptide as described hereinadditionally differs in 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14,15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32,33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50,51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65 amino acidsfrom the amino acid sequence of its parent polypeptide, theseadditionally different amino acids are not chosen from the positionscorresponding to positions 90, 103, 142, 335, 485 and 606 of thepolypeptide of SEQ ID NO: 2.

In an embodiment the present application relates to a variantpolypeptide comprising a substitution at a position corresponding toposition 90 of SEQ ID NO: 2, wherein the polypeptide hasbeta-glucosidase activity and the polypeptide has at least 60%, at least65%, at least 70%, at least 71%, at least 72%, at least 73%, at least74%, at least 75%, at least 76%, at least 77%, at least 78%, at least79%, at least 80%, at least 81%, at least 82%, at least 83%, at least84%, at least 85%, at least 86%, at least 87%, at least 88%, at least89%, at least 90%, at least 91%, at least 92%, at least 93%, at least94%, at least 95%, at least 96%, at least 97%, at least 98% or at least99% sequence identity to the amino acid sequence of a parentbeta-glucosidase. In an embodiment the polypeptide comprises asubstitution at position 90 to L. In an embodiment the variantpolypeptide comprises a substitution M90L.

In an embodiment the present application relates to a variantpolypeptide comprising a substitution at a position corresponding toposition 103 of SEQ ID NO: 2, wherein the polypeptide hasbeta-glucosidase activity and the polypeptide has at least 60%, at least65%, at least 70%, at least 71%, at least 72%, at least 73%, at least74%, at least 75%, at least 76%, at least 77%, at least 78%, at least79%, at least 80%, at least 81%, at least 82%, at least 83%, at least84%, at least 85%, at least 86%, at least 87%, at least 88%, at least89%, at least 90%, at least 91%, at least 92%, at least 93%, at least94%, at least 95%, at least 96%, at least 97%, at least 98% or at least99% sequence identity to the amino acid sequence of a parentbeta-glucosidase. In an embodiment the polypeptide comprises asubstitution at position 103 to A. In an embodiment the variantpolypeptide comprises a substitution N103A.

In an embodiment the present application relates to a variantpolypeptide comprising a substitution at a position corresponding toposition 142 of SEQ ID NO: 2, wherein the polypeptide hasbeta-glucosidase activity and the polypeptide has at least 60%, at least65%, at least 70%, at least 71%, at least 72%, at least 73%, at least74%, at least 75%, at least 76%, at least 77%, at least 78%, at least79%, at least 80%, at least 81%, at least 82%, at least 83%, at least84%, at least 85%, at least 86%, at least 87%, at least 88%, at least89%, at least 90%, at least 91%, at least 92%, at least 93%, at least94%, at least 95%, at least 96%, at least 97%, at least 98% or at least99% sequence identity to the amino acid sequence of a parentbeta-glucosidase. In an embodiment the polypeptide comprises asubstitution at position 142 to S. In an embodiment the variantpolypeptide comprises a substitution G142S.

In an embodiment the present application relates to a variantpolypeptide comprising a substitution at a position corresponding toposition 335 of SEQ ID NO: 2, wherein the polypeptide hasbeta-glucosidase activity and the polypeptide has at least 60%, at least65%, at least 70%, at least 71%, at least 72%, at least 73%, at least74%, at least 75%, at least 76%, at least 77%, at least 78%, at least79%, at least 80%, at least 81%, at least 82%, at least 83%, at least84%, at least 85%, at least 86%, at least 87%, at least 88%, at least89%, at least 90%, at least 91%, at least 92%, at least 93%, at least94%, at least 95%, at least 96%, at least 97%, at least 98% or at least99% sequence identity to the amino acid sequence of a parentbeta-glucosidase. In an embodiment the polypeptide comprises asubstitution at position 335 to V. In an embodiment the variantpolypeptide comprises a substitution M335V.

In an embodiment the present application relates to a variantpolypeptide comprising a substitution at a position corresponding toposition 485 of SEQ ID NO: 2, wherein the polypeptide hasbeta-glucosidase activity and the polypeptide has at least 60%, at least65%, at least 70%, at least 71%, at least 72%, at least 73%, at least74%, at least 75%, at least 76%, at least 77%, at least 78%, at least79%, at least 80%, at least 81%, at least 82%, at least 83%, at least84%, at least 85%, at least 86%, at least 87%, at least 88%, at least89%, at least 90%, at least 91%, at least 92%, at least 93%, at least94%, at least 95%, at least 96%, at least 97%, at least 98% or at least99% sequence identity to the amino acid sequence of a parentbeta-glucosidase. In an embodiment the polypeptide comprises asubstitution at position 485 to I. In an embodiment the variantpolypeptide comprises a substitution M485I.

In an embodiment the present application relates to a variantpolypeptide comprising a substitution at a position corresponding toposition 606 of SEQ ID NO: 2, wherein the polypeptide hasbeta-glucosidase activity and the polypeptide has at least 60%, at least65%, at least 70%, at least 71%, at least 72%, at least 73%, at least74%, at least 75%, at least 76%, at least 77%, at least 78%, at least79%, at least 80%, at least 81%, at least 82%, at least 83%, at least84%, at least 85%, at least 86%, at least 87%, at least 88%, at least89%, at least 90%, at least 91%, at least 92%, at least 93%, at least94%, at least 95%, at least 96%, at least 97%, at least 98% or at least99% sequence identity to the amino acid sequence of a parentbeta-glucosidase. In an embodiment the polypeptide comprises asubstitution at position 606 to A. In an embodiment the variantpolypeptide comprises a substitution L606A.

In an embodiment the variant polypeptide comprises less than 100%sequence identity to the amino acid sequence of a parentbeta-glucosidase. In an embodiment the amino acid sequence of the parentbeta-glucosidase comprises SEQ ID NO: 2. In an embodiment the amino acidsequence of the parent beta-glucosidase consists of SEQ ID NO: 2.

The present application also relates to a variant polypeptide comprisinga substitution at position 90 of SEQ ID NO: 4. In an embodiment thevariant polypeptide comprises a substitution at position 90 of SEQ IDNO: 4 to L. In an embodiment the variant polypeptide comprises thesubstitution M90L in SEQ ID NO: 4. This variant polypeptide is anexample of a variant polypeptide that comprises a substitution at aposition corresponding to position 90 of SEQ ID NO: 2.

The present application also relates to a variant polypeptide comprisinga substitution at position 103 of SEQ ID NO: 4. In an embodiment thevariant polypeptide comprises a substitution at position 103 of SEQ IDNO: 4 to A. In an embodiment the variant polypeptide comprises thesubstitution N103A in SEQ ID NO: 4. This variant polypeptide is anexample of a variant polypeptide that comprises a substitution at aposition corresponding to position 103 of SEQ ID NO: 2.

The present application also relates to a variant polypeptide comprisinga substitution at position 142 of SEQ ID NO: 4. In an embodiment thevariant polypeptide comprises a substitution at position 142 of SEQ IDNO: 4 to S. In an embodiment the variant polypeptide comprises thesubstitution G142S in SEQ ID NO: 4. This variant polypeptide is anexample of a variant polypeptide that comprises a substitution at aposition corresponding to position 142 of SEQ ID NO: 2.

The present application also relates to a variant polypeptide comprisinga substitution at position 335 of SEQ ID NO: 4. In an embodiment thevariant polypeptide comprises a substitution at position 335 of SEQ IDNO: 4 to V. In an embodiment the variant polypeptide comprises thesubstitution M335V in SEQ ID NO: 4. This variant polypeptide is anexample of a variant polypeptide that comprises a substitution at aposition corresponding to position 335 of SEQ ID NO: 2.

The present application also relates to a variant polypeptide comprisinga substitution at position 485 of SEQ ID NO: 4. In an embodiment thevariant polypeptide comprises a substitution at position 485 of SEQ IDNO: 4 to I. In an embodiment the variant polypeptide comprises thesubstitution M485I in SEQ ID NO: 4. This variant polypeptide is anexample of a variant polypeptide that comprises a substitution at aposition corresponding to position 485 of SEQ ID NO: 2.

The present application also relates to a variant polypeptide comprisinga substitution at position 607 of SEQ ID NO: 4. In an embodiment thevariant polypeptide comprises a substitution at position 607 of SEQ IDNO: 4 to A. In an embodiment the variant polypeptide comprises thesubstitution L607A in SEQ ID NO: 4. This variant polypeptide is anexample of a variant polypeptide that comprises a substitution at aposition corresponding to position 606 of SEQ ID NO: 2.

The present application also relates to a variant polypeptide comprisinga substitution at one or more positions corresponding to positions 90,103, 142, 335, 485 and 607 of the polypeptide of SEQ ID NO: 4. In anembodiment the variant polypeptide has beta-glucosidase activity.

The present application relates to a variant polypeptide as describedherein, which is a variant of a parent polypeptide which hasbeta-glucosidase activity and which comprises at least 60%, at least65%, at least 70%, at least 71%, at least 72%, at least 73%, at least74%, at least 75%, at least 76%, at least 77%, at least 78%, at least79%, at least 80%, at least 81%, at least 82%, at least 83%, at least84%, at least 85%, at least 86%, at least 87%, at least 88%, at least89%, at least 90%, at least 91%, at least 92%, at least 93%, at least94%, at least 95%, at least 96%, at least 97%, at least 98% or at least99% sequence identity to the polypeptide of SEQ ID NO: 4.

The present application relates to a variant polypeptide as describedherein, which comprises at least 60%, at least 65%, at least 70%, atleast 71%, at least 72%, at least 73%, at least 74%, at least 75%, atleast 76%, at least 77%, at least 78%, at least 79%, at least 80%, atleast 81%, at least 82%, at least 83%, at least 84%, at least 85%, atleast 86%, at least 87%, at least 88%, at least 89%, at least 90%, atleast 91%, at least 92%, at least 93%, at least 94%, at least 95%, atleast 96%, at least 97%, at least 98% or at least 99% sequence identityto the polypeptide of SEQ ID NO: 4.

The present application relates to a variant polypeptide as describedherein, wherein the position corresponding to position 90 of thepolypeptide of SEQ ID NO: 4 is substituted to L. The present applicationrelates to a variant polypeptide as described herein, wherein theposition corresponding to position 103 of the polypeptide of SEQ ID NO:4 is substituted to A. The present application relates to a variantpolypeptide as described herein, wherein the position corresponding toposition 142 of the polypeptide of SEQ ID NO: 4 is substituted to S. Thepresent application relates to a variant polypeptide as describedherein, wherein the position corresponding to position 335 of thepolypeptide of SEQ ID NO: 4 is substituted to V. The present applicationrelates to a variant polypeptide as described herein, wherein theposition corresponding to position 485 of the polypeptide of SEQ ID NO:4 is substituted to I. The present application relates to a variantpolypeptide as described herein, wherein the position corresponding toposition 607 of the polypeptide of SEQ ID NO: 4 is substituted to A. Thepresent application also relates to a variant polypeptide as describedherein, which comprises one or more of these substitutions. For example,the present application relates to a variant polypeptide as describedherein, wherein the position corresponding to position 90 of thepolypeptide of SEQ ID NO: 4 is substituted to L, the positioncorresponding to position 335 of the polypeptide of SEQ ID NO: 4 issubstituted to V and the position corresponding to position 485 of thepolypeptide of SEQ ID NO: 4 is substituted to I.

The present application relates to a variant polypeptide as describedherein, which comprises substitution M90L. The present applicationrelates to a variant polypeptide as described herein, which comprisessubstitution N103A. The present application relates to a variantpolypeptide as described herein, which comprises substitution G142S. Thepresent application relates to a variant polypeptide as describedherein, which comprises substitution M335V. The present applicationrelates to a variant polypeptide as described herein, which comprisessubstitution M485I. The present application relates to a variantpolypeptide as described herein, which comprises substitution L607A. Thepresent application also relates to a variant polypeptide as describedherein, which comprises one or more of these substitutions. For example,the present application relates to a variant polypeptide as describedherein, which comprises substitution M90L, M335V and M485I.

The present application relates to a variant polypeptide as describedherein, which additionally differs in 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11,12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29,30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47,48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65amino acids from the amino acid sequence of its parent polypeptide. Inan embodiment the parent polypeptide has beta-glucosidase activity andcomprises at least 60%, at least 65%, at least 70%, at least 71%, atleast 72%, at least 73%, at least 74%, at least 75%, at least 76%, atleast 77%, at least 78%, at least 79%, at least 80%, at least 81%, atleast 82%, at least 83%, at least 84%, at least 85%, at least 86%, atleast 87%, at least 88%, at least 89%, at least 90%, at least 91%, atleast 92%, at least 93%, at least 94%, at least 95%, at least 96%, atleast 97%, at least 98% or at least 99% sequence identity to thepolypeptide of SEQ ID NO: 4.

In an embodiment the parent beta-glucosidase is selected from the groupconsisting of the polypeptide comprising the amino acid sequence of SEQID NO: 2; the polypeptide comprising the amino acid sequence of SEQ IDNO: 4; a beta-glucosidase from Aspergillus, such as Aspergillus oryzae,such as the one disclosed in WO 02/095014; the fusion protein havingbeta-glucosidase activity disclosed in WO 2008/057637; abeta-glucosidase from Aspergillus, such as Aspergillus fumigatus, suchas the one disclosed as SEQ ID NO:2 in WO 2005/047499 or SEQ ID NO:5 inWO 2014/130812; an Aspergillus fumigatus beta-glucosidase variant, suchas one disclosed in WO 2012/044915; a beta-glucosidase with thefollowing substitutions: F100D, S283G, N456E, F512Y (using SEQ ID NO: 5in WO 2014/130812 for numbering); a beta-glucosidase from Aspergillussuch as Aspergillus aculeatus, Aspergillus niger or Aspergillus kawachi;a beta-glucosidase from Penicillium, such as Penicillium brasilianumdisclosed as SEQ ID NO:2 in WO 2007/019442; a beta-glucosidase fromTrichoderma, such as Trichoderma reesei, such as ones described in U.S.Pat. Nos. 6,022,725, 6,982,159, 7,045,332, 7,005,289, US 2006/0258554,US 2004/0102619; a beta-glucosidase from Thielavia terrestris (see WO2011/035029); and a beta-glucosidase from Trichophaea saccata (see WO2007/019442). In an embodiment the parent beta-glucosidase may even be abacterial beta-glucosidase.

In an embodiment a variant polypeptide as described herein is lesssensitive to glucose inhibition than the parent polypeptide (e.g.wild-type polypeptide) and thus has a higher glucose tolerance than theparent polypeptide (e.g. wild-type polypeptide). With “having a higherglucose tolerance” is meant that a variant polypeptide as describedherein has a higher catalytic activity in the presence of glucosecompared to the parent polypeptide (e.g. wild-type polypeptide). Awild-type polypeptide is for example the polypeptide comprising theamino acid sequence of SEQ ID NO: 2 or the polypeptide comprising theamino acid sequence of SEQ ID NO: 4.

In an embodiment a variant polypeptide as described herein has a higherratio of remaining activity than the parent polypeptide (e.g. wild-typepolypeptide). Ratio of remaining activity as used herein is defined as:(hydrolyzed cellobiose in presence of 20 g/l glucose)/(hydrolyzedcellobiose without added glucose). The hydrolysis of cellobiose can bemeasured as described in the examples section. In an embodiment thevariant polypeptide as described herein has a ratio of remainingactivity that is at least 5% higher than the ratio of remaining activityof the parent polypeptide (e.g. wild-type polypeptide). In an embodimentthe variant polypeptide as described herein has a ratio of remainingactivity that is at least 10% higher, at least 15% higher, at least 20%higher, at least 25% higher, at least 30% higher, at least 35% higher,at least 40% higher, at least 45% higher, at least 50% higher, at least55% higher, at least 60% higher, at least 65% higher, at least 70%higher, at least 75% higher, at least 80% higher, at least 85% higher,at least 90% higher, at least 95% higher, at least 100% higher, at least125% higher, at least 150% higher, at least 175% higher, at least 200%higher, at least 225% higher, at least 250% higher, at least 275%higher, at least 300% higher, at least 350% higher, at least 400%higher, at least 450% higher, at least 500% higher than the ratio ofremaining activity of the parent polypeptide (e.g. wild-typepolypeptide). In an embodiment the variant polypeptide as describedherein has a ratio of remaining activity that is between 5% and 500%higher than the ratio of remaining activity of the parent polypeptide(e.g. wild-type polypeptide). Preferably, the variant polypeptide asdescribed herein has a ratio of remaining activity that is between 5%and 400% higher than the ratio of remaining activity of the parentpolypeptide (e.g. wild-type polypeptide). Preferably, the variantpolypeptide as described herein has a ratio of remaining activity thatis between 5% and 300% higher than the ratio of remaining activity ofthe parent polypeptide (e.g. wild-type polypeptide). A wild-typepolypeptide is for example the polypeptide comprising the amino acidsequence of SEQ ID NO: 2 or the polypeptide comprising the amino acidsequence of SEQ ID NO: 4.

The present application provides novel polypeptides, e.g. enzymes, whichhave the ability to modify, for example degrade, a carbohydratematerial. A carbohydrate material is a material which comprises,consists of, or substantially consists of one or more carbohydrates. Thepresent application also provides polynucleotides encoding thepolypeptides. In an embodiment a variant polypeptide as described hereinis isolated. In an embodiment the variant polypeptide as describedherein is an enzyme. In an embodiment the variant polypeptide asdescribed herein is a carbohydrate degrading enzyme. In an embodimentthe variant polypeptide as described herein is a carbohydratehydrolysing enzyme. In an embodiment the variant polypeptide asdescribed herein comprises beta-glucosidase activity.

In an embodiment the variant polypeptide as described herein comprisesadvantageously one or more additional substitutions, wherein the one ormore additional substitutions are preferably giving an additional effectwhich may even further improve the advantageous property of the variantpolypeptide as described herein or may give another advantageousproperty to the variant polypeptide as described herein.

In an embodiment the variant polypeptide as described herein hasbeta-glucosidase activity. In an embodiment the variant polypeptide asdescribed herein is a beta-glucosidase. In an embodiment the variantpolypeptide as described herein is a GH3 beta-glucosidase.

A beta-glucosidase (EC 3.2.1.21) is any polypeptide which is capable ofcatalysing the hydrolysis of terminal, non-reducing beta-D-glucoseresidues with release of beta-D-glucose. Such a polypeptide may have awide specificity for beta-D-glucosides and may also hydrolyze one ormore of the following: a beta-D-galactoside, an alpha-L-arabinoside, abeta-D-xyloside or a beta-D-fucoside. This enzyme may also be referredto as amygdalase, beta-D-glucoside glucohydrolase, cellobiase orgentobiase.

By “variant polypeptide comprising a substitution at a positioncorresponding to position X of the polypeptide of SEQ ID NO: Y” is meantthe position X determined from an amino acid sequence alignment of theamino acid sequence of the variant polypeptide with the polypeptide ofSEQ ID NO: Y. This means that the specific substitution claimed withregard to the position X in the amino acid sequence of SEQ ID NO: Y, maybe found on a different position in the amino acid sequence of thevariant polypeptide. For example, the substitution to A on position 606of the amino acid sequence of SEQ ID NO: 2, may have a differentposition in the amino acid sequence of a variant polypeptide. Forexample, the substitution L606A in the amino acid sequence of SEQ ID NO:2 corresponds with the mutation L607A in the amino acid sequence of SEQID NO: 4. In addition, the original amino acid in the parent of thevariant polypeptide may differ from the original amino acid in the aminoacid sequence of SEQ ID NO: 2. The amino acid at a position in theparent of the variant polypeptide that corresponds to position 90 in theamino acid sequence of SEQ ID NO: 2 can differ from the M as found onposition 90 in the amino acid sequence of SEQ ID NO: 2. It could be anyamino acid (except L). The position and type of amino acid to besubstituted in parent polypeptides can be found through alignment of theamino acid sequences of the parent polypeptides with SEQ ID NO: 2. Thealignment can be made using the Clustal Omega computer program (ClustalOmega computer program is a multiple sequence alignment program thatuses seeded guide trees and HMM profile-profile techniques to generatealignments www.ebi.ac.uk/Tools/msa/clustalo).

In an embodiment the one or more substitutions as described hereinincrease thermostability of a variant polypeptide as compared to itsparent polypeptide. In an embodiment the one or more substitutions asdescribed herein increase glucose tolerance of a variant polypeptide ascompared to its parent polypeptide. In an embodiment the one or moresubstitutions as described herein increase thermostability and increaseglucose tolerance of a variant polypeptide as compared to its parentpolypeptide.

As described above, the variant polypeptide as described herein ispreferably a polypeptide such as an enzyme, more preferably is acarbohydrate degrading enzyme and/or carbohydrate hydrolysing enzyme andmost preferably comprises beta-glucosidase activity. A variantpolypeptide as described herein may have one or more alternative and/oradditional activities, for example, one of the other oxidoreductase,transferase, hydrolase, lyase, isomerase or ligase activities mentionedherein.

The application provides the use of a variant polypeptide as describedherein and compositions comprising the variant polypeptide as describedherein in industrial methods as described in more detail herein.

According to a preferred embodiment the variant polypeptide as describedherein is a “thermostable” polypeptide. In another preferred embodimentthe polynucleotide as described herein encodes a variant polypeptide asdescribed herein with a high thermostability that is more stable underlignocellulosic feedstock hydrolysis conditions. Herein, a“thermostable” polypeptide means that the polypeptide has a higherresidual catalytic activity after a heat-shock temperature treatment ascompared to a polypeptide with lower thermostability (e.g. a polypeptidenot having the respective substitution(s), i.e. its parent polypeptide).A higher stability under process conditions can be identified bydetermining the residual catalytic activity of the polypeptide after thehydrolysis reaction time, for example 72 hours.

According to a preferred embodiment the variant polypeptide as describedherein has a pH optimum between pH 2 and pH 8. Preferably, thepolypeptide has a pH optimum of 6 or lower, 5 or lower, 4.5 or lower, 4or lower, or even 3.5 or lower. Preferably, the polypeptide has a pHoptimum of 2 or higher, preferably 2.5 or higher. Preferably, thepolypeptide has a pH optimum of 3 to 5. The pH optimum is the pH duringhydrolysis at which the polypeptide has optimum activity when measuredduring a certain period of time.

Polynucleotide Sequence

The application also relates to a polynucleotide which encodes a variantpolypeptide as described herein. In an embodiment the polynucleotide asdescribed herein is isolated.

In an embodiment the polynucleotide as described herein has at least60%, at least 61%, at least 62%, at least 63%, at least 64%, at least65%, at least 66%, at least 67%, at least 68%, at least 69%, at least70%, at least 71%, at least 72%, at least 73%, at least 74%, at least75%, at least 76%, at least 77%, at least 78%, at least 79%, at least80%, at least 81%, at least 82%, at least 83%, at least 84%, at least85%, at least 86%, at least 87%, at least 88%, at least 89%, at least90%, at least 91%, at least 92%, at least 93%, at least 94%, at least95%, at least 96%, at least 97%, at least 98%, at least 99% sequenceidentity to the nucleotide sequence of SEQ ID NO: 1.

In an embodiment the polynucleotide as described herein has at least60%, at least 61%, at least 62%, at least 63%, at least 64%, at least65%, at least 66%, at least 67%, at least 68%, at least 69%, at least70%, at least 71%, at least 72%, at least 73%, at least 74%, at least75%, at least 76%, at least 77%, at least 78%, at least 79%, at least80%, at least 81%, at least 82%, at least 83%, at least 84%, at least85%, at least 86%, at least 87%, at least 88%, at least 89%, at least90%, at least 91%, at least 92%, at least 93%, at least 94%, at least95%, at least 96%, at least 97%, at least 98%, at least 99% sequenceidentity to the nucleotide sequence of SEQ ID NO: 3.

In an embodiment the polynucleotide as described herein has less than100% sequence identity to the nucleotide sequence of SEQ ID NO: 1. In anembodiment the polynucleotide as described herein has less than 100%sequence identity to the nucleotide sequence of SEQ ID NO: 3.

A polynucleotide as described herein can be isolated using standardmolecular biology techniques and the sequence information providedherein. For example, by using standard hybridization and cloningtechniques as described in Sambrook, J., Fritsh, E. F., and Maniatis, T.Molecular Cloning: A Laboratory Manual. 2nd, ed., Cold Spring HarborLaboratory, Cold Spring Harbor Laboratory Press, Cold Spring Harbor,N.Y., 1989.

Moreover, a polynucleotide may be isolated by the polymerase chainreaction (PCR) using synthetic oligonucleotide primers designed basedupon the sequence information contained in the sequence of thepolynucleotide.

A polynucleotide as described herein can be amplified using cDNA, mRNAor alternatively, genomic DNA, as a template and appropriateoligonucleotide primers according to standard PCR amplificationtechniques. The polynucleotide so amplified can be cloned into anappropriate vector and characterized by DNA sequence analysis.

Furthermore, oligonucleotides corresponding to or hybridizing to anucleotide sequence as described herein can be prepared by standardsynthetic techniques, e.g. using an automated DNA synthesizer.

A polynucleotide which is complementary to a nucleotide sequence is onewhich is sufficiently complementary to the other nucleotide sequencesuch that it can hybridize to the other nucleotide sequence therebyforming a stable duplex. The term “cDNA” (complementary DNA) is definedherein as a DNA molecule which can be prepared by reverse transcriptionfrom a mRNA molecule. cDNA derived from mRNA only contains codingsequences and can be directly translated into the correspondingpolypeptide product. The term “complementary strand” can be usedinterchangeably with the term “complement”. The complement of anucleotide strand can be the complement of a coding strand or thecomplement of a non-coding strand. When referring to double-strandedpolynucleotides, the complement of a polynucleotide encoding apolypeptide refers to the complementary strand of the strand encodingthe amino acid sequence or to any polynucleotide containing the same.

As used herein, the term “hybridization” means the pairing ofsubstantially complementary strands of oligomeric compounds. Onemechanism of pairing involves hydrogen bonding, which may beWatson-Crick, Hoogsteen or reversed Hoogsteen hydrogen bonding, betweencomplementary nucleotide bases (nucleotides) of the strands ofoligomeric compounds. For example, adenine and thymine are complementarynucleic acids which pair through the formation of hydrogen bonds.Hybridization can occur under varying circumstances. “Stringencyhybridization” or “hybridizes under low stringency, medium stringency,high stringency, or very high stringency conditions” is used herein todescribe conditions for hybridization and washing, more specificallyconditions under which an oligomeric compound will hybridize to itstarget sequence, but to a minimal number of other sequences. So, theoligomeric compound will hybridize to the target sequence to adetectably greater degree than to other sequences. Guidance forperforming hybridization reactions can be found in Current Protocols inMolecular Biology, John Wiley & Sons, N.Y. (1989), 6.3.1-6:3.6. Aqueousand non-aqueous methods are described in that reference and either canbe used. Stringency conditions are sequence-dependent and will bedifferent in different circumstances. Generally, stringency conditionsare selected to be about 5° C. lower than the thermal melting point (Tm)for the oligomeric compound at a defined ionic strength and pH. The Tmis the temperature (under defined ionic strength and pH) at which 50% ofan oligomeric compound hybridizes to a perfectly matched probe.Stringency conditions may also be achieved with the addition ofdestabilizing agents such as formamide.

Examples of specific hybridization conditions are as follows: 1) lowstringency hybridization conditions in 6× sodium chloride/sodium citrate(SSC) at about 45° C., followed by two washes in 0.2×SSC, 0.1% SDS atleast at 50° C. (the temperature of the washes can be increased to 55°C. for low stringency conditions); 2) medium stringency hybridizationconditions in 6×SSC at about 45° C., followed by one or more washes in0.2× SSC, 0.1% SDS at 60° C.; 3) high stringency hybridizationconditions in 6×SSC at about 45° C., followed by one or more washes in0.2×SSC, 0.1% SDS at 65° C.; and 4) very high stringency hybridizationconditions are 0.5M sodium phosphate, 7% SDS at 65° C., followed by oneor more washes at 0.2×SSC, 1% SDS at 65° C.

In general, high stringency conditions, such as high hybridizationtemperature and optionally low salt concentrations, permit onlyhybridization between sequences that are highly similar, whereas lowstringency conditions, such as low hybridization temperature andoptionally high salt concentrations, allow hybridization when thesequences are less similar.

One aspect of the application pertains to isolated polynucleotides thatencode a variant polypeptide as described herein as well aspolynucleotides sufficient for use as hybridization probes to identifypolynucleotides encoding a variant polypeptide as described herein.

The term “naturally-occurring” as used herein refers to methods, events,or things that occur in their relevant form in nature. By contrast, “notnaturally-occurring” refers to methods, events, or things whoseexistence or form involves the hand of man. Generally, the term“naturally-occurring” with regard to polypeptides or polynucleotides canbe used interchangeable with the term “wild-type” or “native”. It refersto polypeptide or polynucleotides encoding a polypeptide, having anamino acid sequence or nucleotide sequence, respectively, identical tothat found in nature. Naturally-occurring polypeptides include nativepolypeptides, such as those polypeptides naturally expressed or found ina particular host. Naturally-occurring polynucleotides include nativepolynucleotides such as those polynucleotides naturally found in thegenome of a particular host. Additionally, a sequence that is wild-typeor naturally-occurring may refer to a sequence from which a variant or asynthetic sequence is derived.

In an embodiment the variant polypeptides as described herein and thepolynucleotides as described herein are not naturally-occurring.

As used herein, a “synthetic” molecule is produced by in vitro chemicalor enzymatic synthesis. It includes, but is not limited to,polynucleotides made with optimal codon usage for host organisms ofchoice.

The term “recombinant” when used in reference to a host cell,polynucleotide, polypeptide, nucleic acid construct or vector, indicatesthat the host cell, polynucleotide, polypeptide or vector, has beenmodified by the introduction of a heterologous polynucleotide orpolypeptide or the alteration of a native polynucleotide or polypeptide,or that the host cell is derived from a host cell so modified. Thus, forexample, recombinant host cells express polynucleotides that are notfound within the native (non-recombinant) form of the host cell orexpress native genes that are otherwise abnormally expressed, underexpressed or not expressed at all. The term “recombinant” is synonymouswith “genetically-modified”.

The term “isolated polypeptide” as used herein means a polypeptide thatis removed from at least one component, e.g. other polypeptide material,with which it is naturally associated. Thus, an isolated polypeptide maycontain at most 10%, at most 8%, more preferably at most 6%, morepreferably at most 5%, more preferably at most 4%, more preferably atmost 3%, even more preferably at most 2%, even more preferably at most1% and most preferably at most 0.5% as determined by SDS-PAGE of otherpolypeptide material with which it is natively associated. The isolatedpolypeptide may be free of any other impurities. The isolatedpolypeptide may be at least 50% pure, at least 60% pure, at least 70%pure, at least 75% pure, at least 80% pure, at least 85% pure, at least90% pure, at least 95% pure, at least 96% pure, at least 97% pure, atleast 98% pure, at least 99% pure, at least 99.5% pure, at least 99.9%pure as determined by SDS-PAGE or any other analytical method suitablefor this purpose and known to the person skilled in the art.

An “isolated polynucleotide” or “isolated nucleic acid” is apolynucleotide removed from other polynucleotides with which it isnaturally associated. Thus, an isolated polynucleotide may contain atmost 10%, at most 8%, more preferably at most 6%, more preferably atmost 5%, more preferably at most 4%, more preferably at most 3%, evenmore preferably at most 2%, even more preferably at most 1% and mostpreferably at most 0.5% by weight of other polynucleotide material withwhich it is naturally associated. The isolated polynucleotide may befree of any other impurities. The isolated polynucleotide may be atleast 50% pure, at least 60% pure, at least 70% pure, at least 75% pure,at least 80% pure, at least 85% pure, at least 90% pure, or at least 95%pure, at least 96% pure, at least 97% pure, at least 98% pure, at least99% pure, at least 99.5% pure, at least 99.9% pure by weight.

The term “substantially pure” with regard to polypeptides refers to apolypeptide preparation which contains at the most 50% by weight ofother polypeptide material. The polypeptides disclosed herein arepreferably in a substantially pure form. In particular, it is preferredthat the polypeptides disclosed herein are in “essentially pure form”,i.e. that the polypeptide preparation is essentially free of otherpolypeptide material. Optionally, the polypeptide may also beessentially free of non-polypeptide material such as nucleic acids,lipids, media components, and the like. Herein, the term “substantiallypure polypeptide” is synonymous with the terms “isolated polypeptide”and “polypeptide in isolated form”. The term “substantially pure” withregard to polynucleotide refers to a polynucleotide preparation whichcontains at the most 50% by weight of other polynucleotide material. Thepolynucleotides disclosed herein are preferably in a substantially pureform. In particular, it is preferred that the polynucleotide disclosedherein are in “essentially pure form”, i.e. that the polynucleotidepreparation is essentially free of other polynucleotide material.Optionally, the polynucleotide may also be essentially free ofnon-polynucleotide material such as polypeptides, lipids, mediacomponents, and the like. Herein, the term “substantially purepolynucleotide” is synonymous with the terms “isolated polynucleotide”and “polynucleotide in isolated form”.

The term “nucleic acid” as used in the present application refers to anucleotide polymer including at least 5 nucleotide units. A nucleic acidrefers to a ribonucleotide polymer (RNA), deoxynucleotide polymer (DNA)or a modified form of either type of nucleic acid or synthetic formthereof or mixed polymers of any of the above. Nucleic acids may includeeither or both naturally-occurring and modified nucleic acids linkedtogether by naturally-occurring and/or non-naturally occurring nucleicacid linkages. The nucleic acid molecules may be modified chemically orbiochemically or may contain non-natural or derivatized nucleic acidbases, as will be readily appreciated by those of skill in the art. Suchmodifications include, for example, labels, methylation, substitution ofone or more of the naturally occurring nucleic acids with an analog,internucleotide modifications such as uncharged linkages (e.g., methylphosphonates, phosphotriesters, phosphoramidates, carbamates, etc.),charged linkages (e.g., phosphorothioates, phosphorodithioates, etc.),pendent moieties (e.g., polypeptides), intercalators (e.g., acridine,psoralen, etc.), chelators, alkylators, and modified linkages (e.g.,alpha anomeric nucleic acids, etc.) The term nucleic acid is alsointended to include any topological conformation, includingsingle-stranded (sense strand and antisense strand), double-stranded,partially duplexed, triplex, hairpinned, circular and padlockedconformations. Also included are synthetic molecules that mimic nucleicacids in their ability to bind to a designated sequence via hydrogenbonding and other chemical interactions. Such molecules are known in theart and include, for example, those in which peptide linkages substitutefor phosphate linkages in the backbone of the molecule. A reference to anucleic acid sequence encompasses its complement unless otherwisespecified. Thus, a reference to a nucleic acid molecule having aparticular sequence should be understood to encompass its complementarystrand, with its complementary sequence. The complementary strand isalso useful, e.g., for antisense therapy, hybridization probes and PCRprimers. The term “nucleic acid”, “nucleic acid molecule” and“polynucleotide” can be used interchangeably herein. The term “nucleicacid sequence” and “nucleotide sequence” can also be usedinterchangeably herein.

A “substitution”, as used herein in relation to polypeptides orpolynucleotides, denotes the replacement of one or more amino acids in apolypeptide sequence or of one or more nucleotides in a nucleotidesequence, respectively, by different amino acids or nucleotides,respectively.

Another embodiment of the application provides an isolatedpolynucleotide which is antisense to a polynucleotide as describedherein, e.g. the coding strand of a polynucleotide as described herein.Also included within the scope of the application are the complementarystrands of the polynucleotides described herein.

Unless otherwise indicated, all nucleotide sequences determined bysequencing a DNA molecule herein were determined using an automated DNAsequencer and all amino acid sequences of polypeptides encoded by DNAmolecules determined herein were predicted by translation of a DNAsequence determined as above. Therefore, as is known in the art for anyDNA sequence determined by this automated approach, any nucleotidesequence determined herein may contain some errors. Nucleotide sequencesdetermined by automation are typically at least about 90% identical,more typically at least about 95% to at least about 99.9% identical tothe actual nucleotide sequence of the sequenced DNA molecule.

The actual sequence can be more precisely determined by other approachesincluding manual DNA sequencing methods well known in the art. As isalso known in the art, a single insertion or deletion in a determinednucleotide sequence compared to the actual sequence will cause a frameshift in translation of the nucleotide sequence such that the predictedamino acid sequence encoded by a determined nucleotide sequence will becompletely different from the amino acid sequence actually encoded bythe sequenced DNA molecule, beginning at the point of such an insertionor deletion.

The term “deletion” as used herein denotes a change in either amino acidor nucleotide sequence in which one or more amino acids or nucleotides,respectively, are absent as compared to the parent, often thenaturally-occurring, amino acid or nucleotide sequence.

The term “insertion”, also known as the term “addition”, denotes achange in an amino acid or nucleotide sequence resulting in the additionof one or more amino acids or nucleotides, respectively, as compared tothe parent, often the naturally-occurring, amino acid or nucleotidesequence.

A person skilled in the art is capable of identifying such erroneouslyidentified bases and knows how to correct for such errors.

A polynucleotide as described herein may encoding only a portion of avariant polypeptide as described herein.

The probe/primer typically comprises a substantially purifiedoligonucleotide which typically comprises a nucleotide sequence thathybridizes preferably under highly stringent conditions to at least fromabout 12 to about 15, preferably from about 18 to about 20, preferablyfrom about 22 to about 25, more preferably about 30, about 35, about 40,about 45, about 50, about 55, about 60, about 65, or about 75 or moreconsecutive nucleotides of a nucleotide sequence.

Probes can be used to detect nucleotide sequences encoding the same orhomologous polypeptides, for instance in other organisms. In preferredembodiments, the probe further comprises a label group attached thereto,e.g. the label group can be a radioisotope, a fluorescent compound, anenzyme, or an enzyme cofactor. Such probes can also be used as part of adiagnostic test kit for identifying cells which express a variantpolypeptide as described herein.

The polynucleotides as described herein may be syntheticpolynucleotides. The synthetic polynucleotides may be optimized in codonuse, preferably according to the methods described in WO 2006/077258and/or PCT/EP2007/055943, which are herein incorporated by reference.PCT/EP2007/055943 addresses codon-pair optimization. Codon-pairoptimization is a method wherein the nucleotide sequences encoding apolypeptide have been modified with respect to their codon usage, inparticular the codon pairs that are used, to obtain improved expressionof the nucleotide sequence encoding the polypeptide and/or improvedproduction of the encoded polypeptide. Codon pairs are defined as a setof two subsequent triplets (codons) in a coding sequence. Those skilledin the art will know that the codon usage needs to be adapted dependingon the host species, possibly resulting in variants with significanthomology deviation from a given nucleotide sequence, but still encodinga variant polypeptide as described herein.

The term “expression” includes any step involved in the production ofthe polypeptide including, but not limited to, transcription,post-transcriptional modification, translation, post-translationalmodification, and secretion.

Nucleic Acid Construct

The application further relates to a nucleic acid construct or vectorcomprising a polynucleotide as described herein. The term “nucleic acidconstruct” is herein referred to as a nucleic acid molecule, eithersingle- or double-stranded, which is isolated from a naturally-occurringgene or which has been modified to contain segments of nucleic acidswhich are combined and juxtaposed in a manner which would not otherwiseexist in nature. The term nucleic acid construct is synonymous with theterm “expression cassette” when the nucleic acid construct contains allthe control sequences required for expression of a coding sequence,wherein said control sequences are operably linked to said codingsequence.

The term “coding sequence” as defined herein is a sequence, which istranscribed into mRNA and translated into a polypeptide. The boundariesof the coding sequence are generally determined by the ATG start codonat the 5′-end of the mRNA and a translation stop codon sequenceterminating the open reading frame at the 3′-end of the mRNA. A codingsequence can include, but is not limited to, DNA, cDNA, and recombinantnucleotide sequences. Preferably, the nucleic acid has high GC content.The GC content herein indicates the number of G and C nucleotides in theconstruct, divided by the total number of nucleotides, expressed in %.The GC content is preferably 56% or more, 57% or more, 58% or more, 59%or more, 60% or more, or in the range of 56-70% or the range of 58-65%.Preferably, the nucleic acid construct comprises a promoter sequence, acoding sequence in operative association with said promoter sequence andcontrol sequences, such as (a) a translational termination sequenceorientated in 5′ towards 3′ direction, and/or (b) a translationalinitiator coding sequence orientated in 5′ towards 3′ direction, and/or(c) a translational initiator sequence

In the context of this application, the term “translational initiatorcoding sequence” is defined as the nucleotides immediately downstream ofthe initiator or start codon of the open reading frame of a codingsequence. The initiator or start codon encodes for the AA methionine.The initiator codon is typically ATG, but may also be any functionalstart codon such as GTG.

In the context of this application, the term “translational terminationsequence” is defined as the nucleotides starting from the translationalstop codon at the 3′ end of the open reading frame or nucleotide codingsequence and oriented in 5′ towards 3′ direction.

In the context of this application, the term “translational initiatorsequence” is defined as the nucleotides immediately upstream of theinitiator or start codon of the open reading frame of a sequence codingfor a polypeptide.

In an embodiment the nucleic acid construct is a vector, such as anexpression vector, wherein the polynucleotide as described herein isoperably linked to at least one control sequence for the expression ofthe polynucleotide in a host cell.

An expression vector comprises a polynucleotide coding for apolypeptide, operably linked to the appropriate control sequences (suchas a promoter, and transcriptional and translational stop signals) forexpression and/or translation in vitro or in a host cell. Certainvectors are capable of directing the expression of genes to which theyare operatively linked. Such vectors are referred to herein as“expression vectors”. In general, expression vectors of utility inrecombinant DNA techniques are often in the form of plasmids.

The expression vector may be any vector (e.g. a plasmid or virus), whichcan be conveniently subjected to recombinant DNA procedures and canbring about the expression of the polynucleotide. The choice of thevector will typically depend on the compatibility of the vector with thecell into which the vector is to be introduced. The vectors may belinear or closed circular plasmids. The vector may be an autonomouslyreplicating vector, i.e. a vector which exists as an extra-chromosomalentity, the replication of which is independent of chromosomalreplication, e.g. a plasmid, an extra-chromosomal element, amini-chromosome, or an artificial chromosome. Alternatively, the vectormay be one which, when introduced into the host cell, is integrated intothe genome and replicated together with the chromosome(s) into which ithas been integrated. The integrative cloning vector may integrate atrandom or at a predetermined target locus in the chromosomes of the hostcell.

The vector system may be a single vector or plasmid or two or morevectors or plasmids, which together contain the total DNA to beintroduced into the genome of the host cell, or a transposon.

The vectors preferably contain one or more selectable markers whichpermit easy selection of transformed cells.

Another aspect of the application pertains to vectors, including cloningand expression vectors, comprising a polynucleotide as described hereinencoding and methods of growing, transforming or transfecting suchvectors in a suitable host cell, for example under conditions in whichexpression of a variant polypeptide as described herein occurs.

As used herein, the term “vector” refers to a nucleic acid moleculecapable of transporting another nucleic acid to which it has beenlinked. Thus, in a further embodiment the application provides a methodof making polynucleotides as described herein by introducing apolynucleotide as described herein into a replicable vector, introducingthe vector into a compatible host cell, and growing the host cell underconditions which bring about replication of the vector. The vector maybe recovered from the host cell. Suitable host cells are describedbelow.

One type of vector is a “plasmid”, which refers to a circular doublestranded DNA loop into which additional DNA segments can be ligated.Another type of vector is a viral vector, wherein additional DNAsegments can be ligated into the viral genome. The terms “plasmid” and“vector” can be used interchangeably herein as the plasmid is the mostcommonly used form of vector. However, the application is intended toinclude such other forms of expression vectors, such as cosmids, viralvectors (e.g. replication defective retroviruses, adenoviruses andadeno-associated viruses) and phage vectors which serve equivalentfunctions.

Vectors as described herein may be used in vitro, for example for theproduction of RNA or used to transfect or transform a host cell.

A vector as described herein may comprise two or more, for examplethree, four or five polynucleotides as described herein, for example foroverexpression.

The recombinant expression vectors as described herein comprise apolynucleotide as described herein in a form suitable for expression ofthe polynucleotide in a host cell, which means that the recombinantexpression vector includes one or more regulatory sequences selected onthe basis of the host cells to be used for expression, which is operablylinked to the nucleotide sequence to be expressed.

The term “operably linked”, “operatively linked” or “in operativeassociation” as used herein refers to two or more nucleotide sequenceelements that are physically linked and are in a functional relationshipwith each other. For instance, a promoter is operably linked to a codingsequence, if the promoter is able to initiate or regulate thetranscription or expression of a coding sequence, in which case thecoding sequence should be understood as being “under the control of” thepromoter. Generally, when two nucleotide sequences are operably linked,they will be in the same orientation and usually also in the samereading frame. They usually will be essentially contiguous, althoughthis may not be required.

A vector or nucleic construct for a given host cell may thus comprisethe following elements operably linked to each other in a consecutiveorder from the 5′-end to the 3′-end relative to the coding strand of thesequence encoding a variant polypeptide as described herein: (1) apromoter sequence capable of directing transcription of the nucleotidesequence encoding the variant polypeptide as described herein in thegiven host cell, (2) optionally, a signal sequence capable of directingsecretion of the polypeptide from the given host cell into a culturemedium, (3) a nucleotide sequence as described herein encoding a matureand preferably active form of the variant polypeptide as describedherein, and preferably also (4) a transcription termination region(terminator) capable of terminating transcription downstream of thenucleotide sequence encoding the variant polypeptide as describedherein.

Downstream of the nucleotide sequence as described herein there may be a3′-untranslated region containing one or more transcription terminationsites (e.g. a terminator). The terminator can, for example, be native tothe nucleotide sequence encoding the polypeptide. However, preferably ayeast terminator is used in yeast host cells and a filamentous fungalterminator is used in filamentous fungal host cells. More preferably,the terminator is endogenous to the host cell (in which the nucleotidesequence encoding the polypeptide is to be expressed). In thetranscribed region, a ribosome binding site for translation may bepresent. The coding portion of the mature transcripts expressed by theconstructs will include a translation initiating AUG at the beginningand a termination codon appropriately positioned at the end of thepolypeptide to be translated.

Enhanced expression of the polynucleotide as described herein may alsobe achieved by the selection of heterologous regulatory regions, e.g.promoter, secretion leader and/or terminator regions, which may serve toincrease expression and, if desired, secretion levels of the variantpolypeptide as described herein from the expression host and/or toprovide for the inducible control of the expression of the variantpolypeptide as described herein.

It will be appreciated by those skilled in the art that the design ofthe expression vector can depend on such factors as the choice of thehost cell to be transformed, the level of expression of the polypeptide,etc.

The vectors, such as expression vectors, as described herein can beintroduced into host cells to produce a variant polypeptide as describedherein. The vectors, such as recombinant expression vectors, asdescribed herein can be designed for expression of the polypeptides inprokaryotic or eukaryotic cells.

The recombinant expression vector can also be transcribed and translatedin vitro, for example using T7 promoter regulatory sequences and T7polymerase.

For most filamentous fungi and yeast, the vector or nucleic acidconstruct is preferably integrated in the genome of the host cell inorder to obtain stable transformants. However, for certain yeasts alsosuitable episomal vectors are available into which the expressionconstruct can be incorporated for stable and high level expression.Examples thereof include vectors derived from the 2p and pKD1 plasmidsof Saccharomyces and Kluyveromyces, respectively, or vectors containingan AMA sequence (e.g. AMA1 from Aspergillus). In case the expressionconstructs are integrated in the host cells genome, the constructs areeither integrated at random loci in the genome or at predeterminedtarget loci using homologous recombination, in which case the targetloci preferably comprise a highly expressed gene.

Accordingly, expression vectors useful in the present applicationinclude chromosomal-, episomal- and virus-derived vectors, e.g. vectorsderived from bacterial plasmids, bacteriophage, yeast episome, yeastchromosomal elements, viruses such as baculoviruses, papova viruses,vaccinia viruses, adenoviruses, fowl pox viruses, pseudorabies virusesand retroviruses, and vectors derived from combinations thereof, such asthose derived from plasmid and bacteriophage genetic elements, such ascosmids and phagemids.

The term “control sequence” or “regulatory sequence” can be usedinterchangeably with the term “expression-regulating nucleic acidsequence”. The term as used herein refers to nucleotide sequencesnecessary for and/or affecting the expression of an operably linkedcoding sequence in a particular host organism or in vitro. When twonucleic acid sequences are operably linked, they usually will be in thesame orientation and also in the same reading frame. They usually willbe essentially contiguous, although this may not be required. Theexpression-regulating nucleic acid sequences, such as inter aliaappropriate transcription initiation, termination, promoter, leader,signal peptide, pro-peptide, prepro-peptide, or enhancer sequences;Shine-Delgarno sequence, repressor or activator sequences; efficient RNAmethoding signals such as splicing and polyadenylation signals;sequences that stabilize cytoplasmic mRNA; sequences that enhancetranslation efficiency (e.g. ribosome binding sites); sequences thatenhance protein stability; and when desired, sequences that enhanceprotein secretion, can be any nucleotide sequence showing activity inthe host organism of choice and can be derived from genes encodingproteins, which are either homologous or heterologous to the hostorganism. Each control sequence may be native or foreign to thenucleotide sequence encoding the polypeptide. When desired, the controlsequence may be provided with linkers for the purpose of introducingspecific restriction sites facilitating ligation of the controlsequences with the coding region of the nucleic acid sequence encoding apolypeptide. Control sequences may be optimized to their specificpurpose.

The control sequence may be an appropriate promoter sequence, anucleotide sequence, which is recognized by a host cell for expressionof the nucleotide sequence. The promoter sequence containstranscriptional control sequences, which mediate the expression of thepolypeptide. The promoter may be any nucleotide sequence, which showstranscriptional activity in the cell including mutant, truncated, andhybrid promoters, and may be obtained from genes encoding extracellularor intracellular polypeptides, either homologous or heterologous to thecell.

The term “promoter” is defined herein as a nucleotide sequence thatbinds RNA polymerase and directs the polymerase to the correctdownstream transcriptional start site of a nucleotide sequence encodinga biological compound to initiate transcription. RNA polymeraseeffectively catalyses the assembly of messenger RNA complementary to theappropriate DNA strand of a coding region. The term “promoter” will alsobe understood to include the 5′-non-coding region (between promoter andtranslation start) for translation after transcription into mRNA,cis-acting transcription control elements such as enhancers, and othernucleotide sequences capable of interacting with transcription factors.The promoter may be any appropriate promoter sequence suitable for aeukaryotic or prokaryotic host cell, which shows transcriptionalactivity, including mutant, truncated, and hybrid promoters, and may beobtained from polynucleotides encoding extracellular or intracellularpolypeptides either homologous (native) or heterologous (foreign) to thecell.

The promoter may be a constitutive or inducible promoter. Preferably,the promoter is an inducible promoter. More preferably the promoter is acarbohydrate inducible promoter. Carbohydrate inducible promoters areknown in the art. In a preferred embodiment the promoter is suitable infilamentous fungi. Such promoters are known in the art. In a preferredembodiment the promoter is a Rasamsonia promoter. Preferably, thepromoter sequence is from a highly expressed gene. Highly expressedgenes are known in the art.

The promoters used in the host cells as described herein may bemodified, if desired, to affect their control characteristics. Suitablepromoters in this context include both constitutive and induciblenatural promoters as well as engineered promoters, which are well-knownto the person skilled in the art.

Transcription of the nucleotide sequence encoding the variantpolypeptides as described herein by higher eukaryotes may be increasedby inserting an enhancer sequence into the vector. Enhancers arecis-acting elements of DNA, usually about from 10 to 300 base pairs,that act to increase transcriptional activity of a promoter in a givenhost cell type. Examples of suitable enhancers are well-known to theperson skilled in the art.

The control sequence may also be a suitable transcription terminatorsequence, a sequence recognized by a host cell to terminatetranscription. The terminator sequence is operably linked to the3′-terminus of the nucleotide sequence encoding the polypeptide. Anyterminator, which is functional in the cell, may be used in the presentapplication. Examples of suitable transcription terminator sequences arewell-known to the person skilled in the art.

The control sequence may also include a suitable leader sequence, anon-translated region of a mRNA which is important for translation bythe host cell. The leader sequence is operably linked to the 5′-terminusof the nucleotide sequence encoding the polypeptide. Any leadersequence, which is functional in the cell, may be used in the presentapplication. Examples of suitable leader sequences are well known to theperson skilled in the art.

The control sequence may also be a polyadenylation sequence, a sequencewhich is operably linked to the 3′-terminus of the nucleotide sequenceand which, when transcribed, is recognized by the host cell as a signalto add polyadenosine residues to transcribed mRNA. Any polyadenylationsequence, which is functional in the cell, may be used in the presentapplication. Examples of suitable polyadenylation sequences arewell-known to the person skilled in the art.

When the variant polypeptide as described herein is to be secreted fromthe host cell into the cultivation medium, an appropriate signalsequence can be added to the polypeptide in order to direct the de novosynthesized polypeptide to the secretion route of the host cell. Theperson skilled in the art knows to select an appropriate signal sequencefor a specific host. The signal sequence may be native to the host cell,or may be foreign to the host cell. As an example, a signal sequencefrom a protein native to the host cell can be used. Preferably, saidnative protein is a highly secreted protein. Examples of suitable signalsequences are well-known to the person skilled in the art.

As an alternative for a signal sequence, the variant polypeptide asdescribed herein can be fused to a secreted carrier protein, or partthereof. Such chimeric construct is directed to the secretion route bymeans of the signal sequence of the carrier protein or part thereof. Inaddition, the carrier protein will provide a stabilizing effect to thepolypeptide as described herein and or may enhance solubility. Suchcarrier protein may be any protein. Preferably, a highly secretedprotein is used as a carrier protein. The carrier protein may be nativeor foreign to the variant polypeptide as described herein. The carrierprotein may be native of may be foreign to the host cell. The carrierprotein and variant polypeptide as described herein may contain aspecific amino acid motif to facilitate isolation of the polypeptide.The variant polypeptide as described herein may be released by a specialreleasing agent. The releasing agent may be a proteolytic enzyme or achemical agent. Examples of suitable carrier proteins are well-known tothe person skilled in the art.

As an alternative for secretion of the variant polypeptide as describedherein into the medium, the variant polypeptide as described herein canbe fused to a localisation sequence to target the variant polypeptide asdescribed herein to a desired cellular compartment, organelle of a cell,or membrane. Such sequences are known to the person skilled in the artand include organelle targeting sequences.

Alternatively, the variant polypeptide as described herein is fused toanother protein that has carbohydrate degrading activity. Optionally,the variant polypeptide as described herein is flanked on the C-terminaland/or the N-terminal side by an amino acid motif that facilitatesidentification, isolation and/or purification.

Homology and Identity

The terms “sequence homology” or “sequence identity” are usedinterchangeably herein. For the purpose of this application, it isdefined here that in order to determine the percentage of sequencehomology or sequence identity of two amino acid sequences or of twonucleotide sequences, the sequences are aligned for optimal comparisonpurposes. In order to optimize the alignment between the two sequences,gaps may be introduced in any of the two sequences that are compared.Such alignment can be carried out over the full-length of the sequencesbeing compared. Alternatively, the alignment may be carried out over ashorter length, for example over about 20, about 50, about 100 or morenucleotides or amino acids. The sequence identity is the percentage ofidentical matches between the two sequences over the reported alignedregion.

A comparison of sequences and determination of percentage of sequenceidentity between two sequences can be accomplished using a mathematicalalgorithm. The skilled person will be aware of the fact that severaldifferent computer programs are available to align two sequences anddetermine the identity between two sequences (Kruskal, J. B. (1983) Anoverview of sequence comparison. In D. Sankoff and J. B. Kruskal, (ed.),Time warps, string edits and macromolecules: the theory and practice ofsequence comparison, pp. 1-44 Addison Wesley). The percent sequenceidentity between two amino acid sequences or between two nucleotidesequences may be determined using the Needleman and Wunsch algorithm forthe alignment of two sequences. (Needleman, S. B. and Wunsch, C. D.(1970) J. Mol. Biol. 48, 443-453). Both amino acid sequences andnucleotide sequences can be aligned by the algorithm. TheNeedleman-Wunsch algorithm has been implemented in the computer programNEEDLE. For the purpose of this application, the NEEDLE program from theEMBOSS package was used (version 2.8.0 or higher, EMBOSS: The EuropeanMolecular Biology Open Software Suite (2000) Rice, P. Longden, I. andBleasby, A. Trends in Genetics 16, (6) pp 276-277,emboss.bioinformatics.nl). For amino acid sequences, EBLOSUM62 is usedfor the substitution matrix. For nucleotide sequence, EDNAFULL is used.The optional parameters used are a gap-open penalty of 10 and a gapextension penalty of 0.5. The skilled person will appreciate that allthese different parameters will yield slightly different results butthat the overall percentage identity of two sequences is notsignificantly altered when using different algorithms.

After alignment by the program NEEDLE as described above, the percentageof sequence identity between a query sequence and a sequence asdescribed herein is calculated as follows: Number of correspondingpositions in the alignment showing an identical amino acid or identicalnucleotide in both sequences divided by the total length of thealignment after subtraction of the total number of gaps in thealignment. The identity defined as herein can be obtained from NEEDLE byusing the NOBRIEF option and is labeled in the output of the program as“longest-identity”.

The nucleotide and amino acid sequences as described herein can furtherbe used as a “query sequence” to perform a search against sequencedatabases to, for example, identify other family members or relatedsequences. Such searches can be performed using the NBLAST and XBLASTprograms (version 2.0) of Altschul, et al. (1990) J. Mol. Biol.215:403-10. BLAST nucleotide searches can be performed with the NBLASTprogram, score=100, word length=12 to obtain nucleotide sequenceshomologous to polynucleotides as described herein. BLAST proteinsearches can be performed with the XBLAST program, score=50, wordlength=3 to obtain amino acid sequences homologous to polypeptides asdescribed herein. To obtain gapped alignments for comparison purposes,Gapped BLAST can be utilized as described in Altschul et al., (1997)Nucleic Acids Res. 25(17): 3389-3402. When utilizing BLAST and GappedBLAST programs, the default parameters of the respective programs (e.g.,XBLAST and NBLAST) can be used.

Host Cells

In an embodiment the host cell comprises a variant polypeptide asdescribed herein, a polynucleotide as described herein or a nucleic acidconstruct or vector as described herein.

The term “host cell” as used herein means any type of cell that issusceptible to transformation, transfection, transduction or the likewith a polynucleotide as described herein or a nucleic acid construct orvector as described herein. It encompasses any progeny of a parent cellthat is not identical to the parent cell due to mutations that occurduring replication. In an embodiment the host cell is a recombinant hostcell.

The variant polypeptides as described herein can be expressed in bothprokaryotic and eukaryotic cells.

A prokaryotic host cell includes, but is not limited to, a bacterialhost cell. The term “bacterial cell” includes both Gram-negative andGram-positive microorganisms. Examples of bacteria include, but are notlimited to, bacteria belonging to the genus Bacillus (e.g. B. subtilis,B. amyloliquefaciens, B. licheniformis, B. puntis, B. megaterium, B.halodurans, B. pumilus), Acinetobacter, Nocardia, Xanthobacter,Escherichia (e.g. E. coli), Streptomyces, Erwinia, Klebsiella, Serratia(e.g. S. marcessans), Pseudomonas (e.g. P. aeruginosa), Salmonella (e.g.S. typhimurium, S. typhi). Bacteria also include, but are not limitedto, photosynthetic bacteria (e.g. green non-sulfur bacteria (e.g.Choroflexus, Chloronema), green sulfur bacteria (e.g. Chlorobium,Pelodictyon), purple sulfur bacteria (e.g. Chromatium), and purplenon-sulfur bacteria (e.g. Rhodospirillum, Rhodobacter, andRhodomicrobium).

An eukaryotic host cell includes, but is not limited to, a yeast hostcell, a nematode host cell, a fungal host cell, an amoeba host cell, anavian host cell, an amphibian host cell, a reptilian host cell, an algalhost cell, a mammalian host cell and an insect host cell.

Suitable host cells are discussed further in Goeddel, Gene ExpressionTechnology: Methods in Enzymology 185, Academic Press, San Diego, Calif.(1990). Representative examples of appropriate host cells are describedbelow. Appropriate culture mediums and conditions for thebelow-described host cells are known in the art.

In a preferred embodiment the host cells are fungal cells, preferablyfilamentous fungal cells, more preferably Rasamsonia cells, mostpreferred Rasamsonia emersonii cells.

“Filamentous fungi” are herein defined as eukaryotic microorganisms thatinclude all filamentous forms of the subdivision Eumycotina and Oomycota(as defined by Hawksworth et al., 1995). Filamentous fungal strainsinclude, but are not limited to, strains of Acremonium, Aspergillus,Agaricus, Aureobasidium, Cryptococcus, Corynascus, Chrysosporium,Filibasidium, Fusarium, Humicola, Magnaporthe, Monascus, Mucor,Myceliophthora, Mortierella, Neocallimastix, Neurospora, Paecilomyces,Penicillium, Piromyces, Phanerochaete Podospora, Pycnoporus, Rhizopus,Schizophyllum, Sordaria, Talaromyces, Rasamsonia, Thermoascus,Thielavia, Tolypocladium, Trametes and Trichoderma. Preferredfilamentous fungal strains that may serve as host cells belong to thespecies Aspergillus niger, Aspergillus oryzae, Aspergillus fumigatus,Penicillium chrysogenum, Penicillium citrinum, Acremonium chrysogenum,Trichoderma reesei, Rasamsonia emersonii (formerly known as Talaromycesemersonii), Aspergillus sojae, Chrysosporium lucknowense, Myceliophtorathermophyla.

Preferred yeast host cells may be selected from the genera:Saccharomyces (e.g. S. cerevisiae, S. bayanus, S. pastorianus, S.carlsbergensis), Kluyveromyces, Candida (e.g. C. revkaufi, C.pulcherrima, C. tropicalis, C. utilis), Pichia (e.g. P. pastoris),Schizosaccharomyces, Hansenula, Kloeckera, Schwanniomyces, and Yarrowia(e.g. Y. lipolytica (formerly classified as Candida lipolytica)).

Examples of insect cells, include, but are not limited to, Drosophila,Spodoptera and Trichoplusa. Examples of nematode cells, include, but arenot limited to, C. elegans cells. Examples of amphibian cells, include,but are not limited to, Xenopus laevis cells). Examples of mammaliancells, include, but are not limited to, NIH3T3, 293, CHO, COS, VERO,0127, BHK, Per-C6, Bowes melanoma and HeLa cells.

In the context of the present application, the “parent host cell” andthe “mutant host cell” may be any type of host cell. The specificembodiments of the mutant host cell are described below. It will beclear to those skilled in the art that embodiments applicable to themutant host cell are as well applicable to the parent host cell, unlessotherwise indicated.

The polynucleotide may be heterologous to the genome of the host cell.The term “heterologous” as used herein refers to nucleotide or aminoacid sequences not naturally occurring in a host cell. In other words,the nucleotide or amino acid sequence is not identical to that naturallyfound in the host cell. As used herein, the term “endogenous” or“homologous” refers to a nucleotide or amino acid sequencenaturally-occurring in a host.

In another embodiment, the application features host cells, e.g.transformed host cells or recombinant host cells that contain a nucleicacid encompassed by the application. A “transformed cell” or“recombinant cell” is a cell into which (or into an ancestor of which)has been introduced, by means of recombinant DNA techniques, apolynucleotide as described herein, a nucleic acid construct asdescribed herein and/or a vector as described herein.

As used herein, the terms “transformed” or “transgenic” with referenceto a cell mean that the cell has a non-native (heterologous) nucleotidesequence integrated into its genome or has an episomal plasmid that ismaintained through multiple generations. The term is synonymous with theterm “recombinant” or “genetically modified”.

A host cell can be chosen that modulates the expression of the insertedsequences or modifies and methods the gene product in a specific,desired fashion. Such modifications (e.g. glycosylation) and methoding(e.g. cleavage) of polypeptide products may facilitate optimalfunctioning of the polypeptides.

Various host cells have characteristic and specific mechanisms forpost-translational methoding and modification of polypeptides and geneproducts. Appropriate cell lines or host systems familiar to those ofskill in the art of molecular biology and/or microbiology can be chosento ensure the desired and correct modification and methoding of theforeign polypeptide expressed. To this end, eukaryotic host cells thatpossess the cellular machinery for proper methoding of the primarytranscript, glycosylation, and phosphorylation of the gene product canbe used. Such host cells are well-known in the art.

A host cell as defined herein is an organism suitable for geneticmanipulation and one which may be cultured at cell densities useful forindustrial production of a target product. A suitable organism may be amicroorganism, for example one which may be maintained in a fermentationdevice. A host cell may be a host cell found in nature or a host cellderived from a parent host cell after genetic manipulation or classicalmutagenesis.

According to an embodiment, when the mutant host cell as describedherein is a filamentous fungal host cell, the mutant host cell maycomprise one or more modifications in its genome such that the mutanthost cell is deficient in the production of at least one productselected from glucoamylase (glaA), acid stable alpha-amylase (amyA),neutral alpha-amylase (amyBI and amyBII), oxalic acid hydrolase (oahA),a toxin, preferably ochratoxin and/or fumonisin, a proteasetranscriptional regulator prtT, PepA, a product encoded by the gene hdfAand/or hdfB, a non-ribosomal peptide synthase npsE if compared to aparent host cell and measured under the same conditions.

Therefore, when the mutant microbial host cell as described herein is afilamentous fungal host cell, the host cell may comprise one or moremodifications in its genome to result in a deficiency in the productionof the major extracellular aspartic protease PepA. For example, the hostcell as described herein may further comprise a disruption of the pepAgene encoding the major extracellular aspartic protease PepA.

When the mutant microbial host cell as described herein is a filamentousfungal host cell, the host cell as described herein may additionallycomprises one or more modifications in its genome to result in adeficiency in the production of the product encoded by the hdfA (Ku70)and/or hdfB (Ku80) gene. For example, the host cell as described hereinmay further comprise a disruption of the hdfA and/or hdfB gene.

When the mutant host cell as described herein is a filamentous fungalhost cell, the host cell as described herein may additionally comprise amodification in its genome which results in the deficiency in theproduction of the non-ribosomal peptide synthase npsE.

Host cells as described herein include plant cells and the applicationtherefore extends to transgenic organisms, such as plants and partsthereof, which contain one or more cells as described herein. The cellsmay heterologous express the variant polypeptide as described herein ormay heterologous contain one or more of the polynucleotides as describedherein. The transgenic (or genetically modified) plant may thereforehave inserted (e.g. stably) into its genome a sequence encoding one ormore of the variant polypeptides as described herein. The transformationof plant cells can be performed using known techniques.

In an embodiment the gene encoding for the endogenous and/or parentbeta-glucosidase is deleted or modified in such a way that theendogenous and/or parent beta-glucosidase polypeptide is no longerproduced by the host cells as described herein. Consequently, instead ofthe endogenous and/or parent beta-glucosidase the variant polypeptide asdescribed herein may be produced by the host cells. In other words, thepresent application also provides a host cell wherein the gene encodingthe beta-glucosidase comprising the amino acid sequence of SEQ ID NO: 2has been deleted or modified in such a way that the beta-glucosidasepolypeptide is no longer produced by the host cells. Such host cells maycomprise the polynucleotide that encodes a variant polypeptide asdescribed herein instead and produce said variant beta-glucosidase.

The present application also provides a host cell wherein the geneencoding the beta-glucosidase comprising the amino acid sequence of SEQID NO: 4 has been deleted or modified in such a way that thebeta-glucosidase polypeptide is no longer produced by the host cells.Such host cells may comprise the polynucleotide that encodes a variantpolypeptide as described herein instead and produce said variantbeta-glucosidase.

Polypeptide Production

The application also relates to a method for producing a variantpolypeptide as described herein, which method comprises the steps of (a)cultivating a host cell as described herein under conditions conduciveto the production of the variant polypeptide as described herein, and(b) optionally, recovering the variant polypeptide as described herein.

The host cells as described herein may be cultured using proceduresknown in the art. For each combination of a promoter and a host cell,culture conditions are available which are conducive to the expressionof the polynucleotide sequence encoding the variant polypeptide asdescribed herein. After reaching the desired cell density or titer ofthe polypeptide, the culture is stopped and the polypeptide is recoveredusing known procedures. Alternatively, the polypeptide may not berecovered by used in the form of a whole fermentation broth, eitherkilled of or not killed of. The broth may comprise other constituentsnext to the variant polypeptide as described herein such as cells orparts thereof, culture medium components, to name just a few.

The fermentation medium can comprise a known culture medium containing acarbon source, a nitrogen source, and an inorganic nutrient sources.Optionally, an inducer may be included.

The selection of the appropriate medium may be based on the choice ofexpression host and/or based on the regulatory requirements of thenucleic acid construct. Such media are known to those skilled in theart. The medium may, if desired, contain additional components favoringthe transformed host cell over other potentially contaminatingmicroorganisms.

The fermentation can be performed over a period of from about 0.5 toabout 30 days. It may be a batch, continuous or fed-batch method,suitably at a temperature in the range of 0 to 100° C. or 0 to 80° C.,for example from 0 to 50° C. and/or at a pH from 2 to 10. Preferredfermentation conditions are a temperature in the range of from 20° C. to45° C. and/or at a pH of from 3 to 9. The appropriate conditions areusually selected based on the choice of the host cell and thepolypeptide to be expressed.

After fermentation, if necessary, the cells can be removed from thefermentation broth by means of centrifugation or filtration. Afterfermentation has stopped or after removal of the cells, the avariantpolypeptide as described herein may then be recovered and, if desired,purified and isolated by conventional means.

The variant polypeptide as described herein can be recovered andpurified from recombinant host cell cultures by methods known in theart. Most preferably, high performance liquid chromatography (“HPLC”) isemployed for purification.

If desired, a host cell as described above may be used to in thepreparation of a variant polypeptide as described herein. Such a methodtypically comprises cultivating a host cell (e.g. transformed ortransfected with an nucleic acid construct as described above) underconditions to provide for expression of a coding sequence encoding thepolypeptide, and optionally recovering the expressed polypeptide.Polynucleotides as described herein can be incorporated into arecombinant replicable vector, e.g. an expression vector. The vector maybe used to replicate the nucleic acid in a compatible host cell. Thus ina further embodiment, the application provides a method of making apolynucleotide as described herein by introducing a polynucleotide asdescribed herein into a replicable vector, introducing the vector into acompatible host cell, and growing the host cell under conditions whichbring about the replication of the vector. The vector may be recoveredfrom the host cell.

Preferably, the polypeptide is produced as a secreted protein in whichcase the nucleotide sequence encoding the polypeptide in the expressionconstruct is operably linked to a nucleotide sequence encoding a signalsequence. Preferably, the signal sequence is native (homologous) to thenucleotide sequence encoding the polypeptide. Alternatively, the signalsequence is foreign (heterologous) to the nucleotide sequence encodingthe polypeptide, in which case the signal sequence is preferablyendogenous to the host cell in which the nucleotide sequence asdescribed herein is expressed.

In an embodiment the variant polypeptides as described herein may beoverexpressed in a host cell compared to the parent host cell in whichthe polypeptide is not overexpressed. Overexpression of a polypeptide isdefined herein as the expression of the polypeptide which results in anactivity of the polypeptide in the host cell being at least 1.1-, atleast 1.25- or at least 1.5-fold the activity of the polypeptide in theparent host cell wherein the polypeptide is not overexpressed.

Preferably, the activity of the polypeptide is at least 2-fold, morepreferably at least 3-fold, more preferably at least 4-fold, morepreferably at least 5-fold, even more preferably at least 10-fold andmost preferably at least 20-fold the activity of the polypeptide in theparent host cell.

Transformation of the host cell may be conducted by any suitable knownmethods, including electroporation methods, particle bombardment ormicro projectile bombardment, protoplast methods and Agrobacteriummediated transformation (AMT).

In order to enhance the amount of copies of the polynucleotide codingfor the polypeptide or coding for a compound involved in the productionby the cell of the polypeptide in the mutated host cell, multipletransformations of the host cell may be required. In this way, theratios of the different polypeptides produced by the host cell may beinfluenced. Also, an expression vector may comprise multiple expressioncassettes to increase the amount of copies of the polynucleotide(s) tobe transformed.

Another way could be to choose different control sequences for thedifferent polynucleotides, which—depending on the choice—may cause ahigher or a lower production of the desired polypeptide(s).

The host cells transformed with the selectable marker can be selectedbased on the presence of the selectable marker.

For stable transfection of mammalian cells, it is known that, dependingupon the expression vector and transfection technique used, only a smallfraction of cells may integrate the foreign polynucleotide into theirgenome. In order to identify and select these integrants, a gene thatencodes a selectable marker (e.g. resistance to antibiotics) isgenerally introduced into the host cells along with the polynucleotideof interest. Preferred selectable markers include, but are not limitedto, those which confer resistance to drugs or which complement a defectin the host cell. The selectable marker may be introduced into the cellon the expression vector as the expression cassette or may be introducedon a separate expression vector.

Preferred selectable markers include, but are not limited to, thosewhich confer resistance to drugs or which complement a defect in thehost cell. Alternatively, specific selection markers can be used such asauxotrophic markers which require corresponding mutant host cells. In apreferred embodiment the selection marker is deleted from thetransformed host cell after introduction of the expression construct, soas to obtain transformed host cells which are free of selection markergenes. As indicated, the expression vectors will preferably containselectable markers. Vectors preferred for use in bacteria are forexample disclosed in WO 2004/074468. Other suitable vectors will bereadily apparent to the skilled artisan.

Compositions

The variant polypeptide as described herein may be comprised in acomposition. Preferably, the composition is enriched in the polypeptide.By “enriched” is meant that the polypeptide in the composition isincreased, for example with at least a factor of 1.1, preferably 1.5,more preferably 2 on protein level compared to the composition withoutthe overexpressed variant polypeptide as described herein. Thecomposition may comprise a variant polypeptide as described herein asthe major enzymatic component, e.g. a mono-component composition.Alternatively, the composition may comprise multiple enzymaticactivities. The polypeptide compositions may be prepared in accordancewith methods known in the art and may be in the form of a liquid or adry composition. For instance, the polypeptide composition may be in theform of a granulate or a microgranulate. The polypeptide to be includedin the composition may be stabilized in accordance with methods known inthe art. The dosage of the composition as described herein and otherconditions under which the composition is used depend on the ultimateuse of the composition.

The application is concerned with a composition comprising (a) a variantpolypeptide as described herein, and (b) a cellulase and/or ahemicellulase and/or a pectinase.

In an embodiment the cellulase is selected from the group consisting oflytic polysaccharide monooxygenase, a cellobiohydrolase I, acellobiohydrolase II, an endo-beta-1,4-glucanase, a beta-glucosidase, abeta-(1,3)(1,4)-glucanase and any combination thereof. In an embodimentthe hemicellulase is selected from the group consisting of anendoxylanase, a beta-xylosidase, an alpha-L-arabinofuranosidase, analpha-D-glucuronidase, an acetyl-xylan esterase, a feruloyl esterase, acoumaroyl esterase, an alpha-galactosidase, a beta-galactosidase, abeta-mannanase, a beta-mannosidase and any combination thereof. Ofcourse, the composition may also comprise more than one cellulase and/ormore than one hemicellulase and/or more than one pectinase. For example,two cellulases, two hemicellulases and one pectinase or five cellulases,one hemicellulose and three pectinases. Any combination is possible.Suitable cellulases and/or hemicellulases and/or pectinases aredescribed herein.

Polypeptides can be produced by different methods and mixed into anoptimal composition or the compositions can be made directly as amixture by one fermentation.

A composition as described herein may comprise one, two or three or moreclasses of cellulase, for example a polypeptide as described herein, anendo-1,4-β-glucanase (EG), an exo-cellobiohydrolase (CBH) and a lyticpolysaccharide monooxygenase (LPMO).

A composition as described herein may comprise a polypeptide which hasthe same enzymatic activity, for example the same type of celluloseand/or hemicellulase and/or pectinase activity as that provided by thevariant polypeptide as described herein.

A composition as described herein may comprise a polypeptide which has adifferent type of cellulase activity and/or hemicellulase activityand/or pectinase activity than that provided by the variant polypeptideas described herein. For example, a composition as described herein maycomprise one type of cellulase and/or hemicellulase activity and/orpectinase activity provided by a variant polypeptide as described hereinand a second type of cellulase and/or hemicellulase activity and/orpectinase activity provided by an additionalcellulose/hemicellulase/pectinase.

Herein, a cellulase is any polypeptide which is capable of degradingand/or hydrolysing cellulose or enhancing the degradation and/orhydrolysis of cellulose. A polypeptide which is capable of degradingcellulose is a polypeptide which is capable of catalysing the method ofbreaking down cellulose into smaller units, either partially, forexample into cellodextrins, or completely into glucose monomers.Degradation will typically take place by a hydrolysis reaction.

Herein, a hemicellulase is any polypeptide which is capable of degradingand/or hydrolysing hemicellulose or enhancing the degradation and/orhydrolysis of hemicellulose. That is to say, a hemicellulase may becapable of degrading one or more of xylan, glucuronoxylan, arabinoxylan,glucomannan and xyloglucan. A polypeptide which is capable of degradinga hemicellulose is a polypeptide which is capable of catalysing themethod of breaking down the hemicellulose into smaller polysaccharides,either partially, for example into oligosaccharides, or completely intosugar monomers, for example hexose or pentose sugar monomers. Ahemicellulase may give rise to a mixed population of oligosaccharidesand sugar monomers. Degradation will typically take place by ahydrolysis reaction.

Herein, a pectinase is any polypeptide which is capable of degradingpectin. A polypeptide which is capable of degrading pectin is apolypeptide which is capable of catalysing the method of breaking downpectin into smaller units, either partially, for example intooligosaccharides, or completely into sugar monomers. A pectinase asdescribed herein may give rise to a mixed population of oligosaccharidesand sugar monomers. Degradation will typically take place by ahydrolysis reaction.

The composition may comprise a cellulase and/or a hemicellulase and/or apectinase from Rasamsonia or a source other than Rasamsonia. They may beused together with one or more Rasamsonia enzymes or they may be usedwithout additional Rasamsonia enzymes being present.

The composition as described herein may comprise a beta-glucosidase. Forexample, the composition as described herein may comprise abeta-glucosidase (BG) from Aspergillus, such as Aspergillus oryzae, suchas the one disclosed in WO 02/095014 or the fusion protein havingbeta-glucosidase activity disclosed in WO 2008/057637, or Aspergillusfumigatus, such as the one disclosed as SEQ ID NO:2 in WO 2005/047499 orSEQ ID NO:5 in WO 2014/130812 or an Aspergillus fumigatusbeta-glucosidase variant, such as one disclosed in WO 2012/044915, suchas one with the following substitutions: F100D, S283G, N456E, F512Y(using SEQ ID NO: 5 in WO 2014/130812 for numbering), or Aspergillusaculeatus, Aspergillus niger or Aspergillus kawachi. In anotherembodiment the beta-glucosidase is derived from Penicillium, such asPenicillium brasilianum disclosed as SEQ ID NO:2 in WO 2007/019442, orfrom Trichoderma, such as Trichoderma reesei, such as ones described inU.S. Pat. Nos. 6,022,725, 6,982,159, 7,045,332, 7,005,289, US2006/0258554 US 2004/0102619. In an embodiment even a bacterialbeta-glucosidase can be used. In another embodiment the beta-glucosidaseis derived from Thielavia terrestris (WO 2011/035029) or Trichophaeasaccata (WO 2007/019442). In a preferred embodiment the enzymecomposition comprises a beta-glucosidase from Rasamsonia, such asRasamsonia emersonii (see WO 2012/000886).

The composition as described herein may comprise an endoglucanase. Forexample, the composition as described herein may comprise anendoglucanase (EG) from Trichoderma, such as Trichoderma reesei; fromHumicola, such as a strain of Humicola insolens; from Aspergillus, suchas Aspergillus aculeatus or Aspergillus kawachfi; from Erwinia, such asErwinia carotovara; from Fusarium, such as Fusarium oxysporum; fromThielavia, such as Thielavia terrestris; from Humicola, such as Humicolagrisea var. thermoidea or Humicola insolens; from Melanocarpus, such asMelanocarpus albomyces; from Neurospora, such as Neurospora crassa; fromMyceliophthora, such as Myceliophthora thermophila; from Cladorrhinum,such as Cladorrhinum foecundissimum and/or from Chrysosporium, such as astrain of Chrysosporium lucknowense. In an embodiment even a bacterialendoglucanase can be used including, but are not limited to,Acidothermus cellulolyticus endoglucanase (see WO 91/05039; WO 93/15186;U.S. Pat. No. 5,275,944; WO 96/02551; U.S. Pat. No. 5,536,655, WO00/70031, WO 05/093050); Thermobifida fusca endoglucanase III (see WO05/093050); and Thermobifida fusca endoglucanase V (see WO 05/093050).In a preferred embodiment the endoglucanase is from Rasamsonia, such asRasamsonia emersonii (see WO 01/70998).

The composition as described herein may comprise a cellobiohydrolase I.For example, the composition as described herein may comprise acellobiohydrolase I from Aspergillus, such as Aspergillus fumigatus,such as the Cel7A CBH I disclosed in SEQ ID NO:6 in WO 2011/057140 orSEQ ID NO:6 in WO 2014/130812, or from Trichoderma, such as Trichodermareesei. In a preferred embodiment the enzyme composition comprises acellobiohydrolase I from Rasamsonia, such as Rasamsonia emersonii (seeWO 2010/122141).

The composition as described herein may comprise a cellobiohydrolase II.For example, the composition as described herein may comprise acellobiohydrolase II from Aspergillus, such as Aspergillus fumigatus,such as the one in SEQ ID NO:7 in WO 2014/130812 or from Trichoderma,such as Trichoderma reesei, or from Thielavia, such as Thielaviaterrestris, such as cellobiohydrolase II CEL6A from Thielaviaterrestris. In a preferred embodiment the enzyme composition comprises acellobiohydrolase II from Rasamsonia, such as Rasamsonia emersonii (seeWO 2011/098580).

For example, the composition as described herein may comprise apolypeptide having cellulolytic enhancing activity such as a lyticpolysaccharide monooxygenase from Thermoascus, such as Thermoascusaurantiacus, such as the one described in WO 2005/074656 as SEQ ID NO:2and SEQ ID NO:1 in WO2014/130812 and in WO 2010/065830; or fromThielavia, such as Thielavia terrestris, such as the one described in WO2005/074647 as SEQ ID NO: 8 or SEQ ID NO:4 in WO2014/130812 and in WO2008/148131, and WO 2011/035027; or from Aspergillus, such asAspergillus fumigatus, such as the one described in WO 2010/138754 asSEQ ID NO:2 or SEQ ID NO: 3 in WO2014/130812; or from Penicillium, suchas Penicillium emersonii, such as the one disclosed as SEQ ID NO:2 in WO2011/041397 or SEQ ID NO:2 in WO2014/130812. Other suitable polypeptideshaving cellulolytic enhancing activity such as a lytic polysaccharidemonooxygenases include, but are not limited to, Trichoderma reesei (seeWO 2007/089290), Myceliophthora thermophila (see WO 2009/085935, WO2009/085859, WO 2009/085864, WO 2009/085868), Penicillium pinophilum(see WO 2011/005867), Thermoascus sp. (see WO 2011/039319), andThermoascus crustaceous (see WO 2011/041504). In a preferred embodiment,the lytic polysaccharide monooxygenase is from Rasamsonia, e.g.Rasamsonia emersonii (see WO 2012/000892). In one aspect, thepolypeptide having cellulolytic enhancing activity such as a lyticpolysaccharide monooxygenase is used in the presence of a solubleactivating divalent metal cation according to WO 2008/151043, e.g.manganese sulfate. In one aspect, the polypeptide having cellulolyticenhancing activity such as a lytic polysaccharide monooxygenase is usedin the presence of a dioxy compound, a bicylic compound, a heterocycliccompound, a nitrogen-containing compound, a quinone compound, asulfur-containing compound, or a liquor obtained from a pretreatedcellulosic material such as pretreated corn stover.

Other cellulolytic enzymes that may be comprised in the composition asdescribed herein are described in WO 98/13465, WO 98/015619, WO98/015633, WO 99/06574, WO 99/10481, WO 99/025847, WO 99/031255, WO2002/101078, WO 2003/027306, WO 2003/052054, WO 2003/052055, WO2003/052056, WO 2003/052057, WO 2003/052118, WO 2004/016760, WO2004/043980, WO 2004/048592, WO 2005/001065, WO 2005/028636, WO2005/093050, WO 2005/093073, WO 2006/074005, WO 2006/117432, WO2007/071818, WO 2007/071820, WO 2008/008070, WO 2008/008793, U.S. Pat.Nos. 5,457,046, 5,648,263, and 5,686,593, to name just a few.

In addition, the composition as described herein may comprise anendoxylanase. Examples of endoxylanases that may be comprised in thecomposition as described herein include, but are not limited to,endoxylanases from Aspergillus aculeatus (see WO 94/21785), Aspergillusfumigatus (see WO 2006/078256), Penicillium pinophilum (see WO2011/041405), Penicillium sp. (see WO 2010/126772), Thielavia terrestrisNRRL 8126 (see WO 2009/079210), and Trichophaea saccata GH10 (see WO2011/057083). In a preferred embodiment the enzyme composition comprisesan endoxylanase from Rasamsonia, such as Rasamsonia emersonii (see WO02/24926).

In addition, the composition as described herein may comprise abeta-xylosidase. Examples of beta-xylosidases that may be comprised inthe composition as described herein include, but are not limited to,beta-xylosidases from Neurospora crassa and Trichoderma reesei. In apreferred embodiment the enzyme composition comprises a beta-xylosidasefrom Rasamsonia, such as Rasamsonia emersonii (see WO 2014/118360).

In addition, the composition as described herein may comprise anacetylxylan esterase. Examples of acetylxylan esterases that may becomprised in the enzyme composition include, but are not limited to,acetylxylan esterases from Aspergillus aculeatus (see WO 2010/108918),Chaetomium globosum, Chaetomium gracile, Humicola insolens DSM 1800 (seeWO 2009/073709), Hypocrea jecorina (see WO 2005/001036), Myceliophterathermophila (see WO 2010/014880), Neurospora crassa, Phaeosphaerianodorum and Thielavia terrestris NRRL 8126 (see WO 2009/042846).Examples of feruloyl esterases (ferulic acid esterases) that may becomprised in the enzyme composition include, but are not limited to,feruloyl esterases form Humicola insolens DSM 1800 (see WO 2009/076122),Neosartorya fischeri, Neurospora crassa, Penicillium aurantiogriseum(see WO 2009/127729), and Thielavia terrestris (see WO 2010/053838 andWO 2010/065448). Examples of arabinofuranosidases that may be comprisedin the enzyme composition include, but are not limited to,arabinofuranosidases from Aspergillus niger, Humicola insolens DSM 1800(see WO 2006/114094 and WO 2009/073383) and M. giganteus (see WO2006/114094). Examples of alpha-glucuronidases that may be comprised inthe enzyme composition include, but are not limited to,alpha-glucuronidases from Aspergillus clavatus, Aspergillus fumigatus,Aspergillus niger, Aspergillus terreus, Humicola insolens (see WO2010/014706), Penicillium aurantiogriseum (see WO 2009/068565) andTrichoderma reesei.

A composition as described herein may comprise one, two, three, fourclasses or more of cellulase, for example one, two, three or four or allof a lytic polysaccharide monooxygenase (LPMO), an endoglucanase (EG),one or two exo-cellobiohydrolases (CBH) and a beta-glucosidase (BG). Anenzyme composition as described herein may comprise two or more of anyof these classes of cellulase.

A composition as described herein may comprise one type of cellulaseactivity and/or hemicellulase activity and/or pectinase activityprovided by a composition as described herein and a second type ofcellulase activity and/or hemicellulase activity and/or pectinaseactivity provided by an additional cellulase/hemicellulase/pectinase.Accordingly, a composition as described herein may comprise anycellulase, for example, a lytic polysaccharide monooxygenase, acellobiohydrolase, an endo-beta-1,4-glucanase, a beta-glucosidase or abeta-(1,3)(1,4)-glucanase.

In an embodiment a composition as described herein comprises a variantpolypeptide as described herein, an endoglucanase, a beta-glucosidase, acellobiohydrolase I, a cellobiohydrolase II, an endoxylanase, abeta-xylosidase, and a lytic polysaccharide monooxygenase.

As used herein, a cellobiohydrolase (EC 3.2.1.91) is any polypeptidewhich is capable of catalyzing the hydrolysis of 1,4-beta-D-glucosidiclinkages in cellulose or cellotetraose, releasing cellobiose from theends of the chains. This enzyme may also be referred to as cellulase1,4-beta-cellobiosidase, 1,4-beta-cellobiohydrolase, 1,4-beta-D-glucancellobiohydrolase, avicelase, exo-1,4-beta-D-glucanase,exocellobiohydrolase or exoglucanase.

As used herein, an endo-beta-1,4-glucanase (EC 3.2.1.4) is anypolypeptide which is capable of catalyzing the endohydrolysis of1,4-beta-D-glucosidic linkages in cellulose, lichenin or cerealbeta-D-glucans. Such a polypeptide may also be capable of hydrolyzing1,4-linkages in beta-D-glucans also containing 1,3-linkages. This enzymemay also be referred to as cellulase, avicelase, beta-1,4-endoglucanhydrolase, beta-1,4-glucanase, carboxymethyl cellulase, celludextrinase,endo-1,4-beta-D-glucanase, endo-1,4-beta-D-glucanohydrolase,endo-1,4-beta-glucanase or endoglucanase.

As used herein, a beta-glucosidase (EC 3.2.1.21) is any polypeptidewhich is capable of catalysing the hydrolysis of terminal, non-reducingbeta-D-glucose residues with release of beta-D-glucose. Such apolypeptide may have a wide specificity for beta-D-glucosides and mayalso hydrolyze one or more of the following: a beta-D-galactoside, analpha-L-arabinoside, a beta-D-xyloside or a beta-D-fucoside. This enzymemay also be referred to as amygdalase, beta-D-glucoside glucohydrolase,cellobiase or gentobiase.

As used herein, a beta-(1,3)(1,4)-glucanase (EC 3.2.1.73) is anypolypeptide which is capable of catalysing the hydrolysis of1,4-beta-D-glucosidic linkages in beta-D-glucans containing 1,3- and1,4-bonds. Such a polypeptide may act on lichenin and cerealbeta-D-glucans, but not on beta-D-glucans containing only 1,3- or1,4-bonds. This enzyme may also be referred to as licheninase,1,3-1,4-beta-D-glucan 4-glucanohydrolase, beta-glucanase,endo-beta-1,3-1,4 glucanase, lichenase or mixed linkage beta-glucanase.An alternative for this type of enzyme is EC 3.2.1.6, which is describedas endo-1,3(4)-beta-glucanase. This type of enzyme hydrolyses 1,3- or1,4-linkages in beta-D-glucanse when the glucose residue whose reducinggroup is involved in the linkage to be hydrolysed is itself substitutedat C-3. Alternative names include endo-1,3-beta-glucanase, laminarinase,1,3-(1,3;1,4)-beta-D-glucan 3 (4) glucanohydrolase. Substrates includelaminarin, lichenin and cereal beta-D-glucans.

A composition as described herein may comprise any hemicellulase, forexample, an endoxylanase, a beta-xylosidase, aalpha-L-arabionofuranosidase, an alpha-D-glucuronidase, an acetyl xylanesterase, a feruloyl esterase, a coumaroyl esterase, analpha-galactosidase, a beta-galactosidase, a beta-mannanase or abeta-mannosidase.

As used herein, an endoxylanase (EC 3.2.1.8) is any polypeptide which iscapable of catalysing the endohydrolysis of 1,4-beta-D-xylosidiclinkages in xylans. This enzyme may also be referred to asendo-1,4-beta-xylanase or 1,4-beta-D-xylan xylanohydrolase.

An alternative is EC 3.2.1.136, a glucuronoarabinoxylan endoxylanase, anenzyme that is able to hydrolyze 1,4-xylosidic linkages inglucuronoarabinoxylans.

As used herein, a beta-xylosidase (EC 3.2.1.37) is any polypeptide whichis capable of catalysing the hydrolysis of 1,4-beta-D-xylans, to removesuccessive D-xylose residues from the non-reducing termini. Such enzymesmay also hydrolyze xylobiose. This enzyme may also be referred to asxylan 1,4-beta-xylosidase, 1,4-beta-D-xylan xylohydrolase,exo-1,4-beta-xylosidase or xylobiase.

As used herein, an alpha-L-arabinofuranosidase (EC 3.2.1.55) is anypolypeptide which is capable of acting on alpha-L-arabinofuranosides,alpha-L-arabinans containing (1,2)- and/or (1,3)- and/or (1,5)-linkages,arabinoxylans and arabinogalactans. This enzyme may also be referred toas alpha-N-arabinofuranosidase, arabinofuranosidase or arabinosidase.

As used herein, an alpha-D-glucuronidase (EC 3.2.1.139) is anypolypeptide which is capable of catalysing a reaction of the followingform: alpha-D-glucuronoside+H(2)O=an alcohol+D-glucuronate. This enzymemay also be referred to as alpha-glucuronidase or alpha-glucosiduronase.These enzymes may also hydrolyse 4-O-methylated glucoronic acid, whichcan also be present as a substituent in xylans. An alternative is EC3.2.1.131: xylan alpha-1,2-glucuronosidase, which catalyses thehydrolysis of alpha-1,2-(4-O-methyl)glucuronosyl links.

As used herein, an acetyl xylan esterase (EC 3.1.1.72) is anypolypeptide which is capable of catalysing the deacetylation of xylansand xylo-oligosaccharides. Such a polypeptide may catalyze thehydrolysis of acetyl groups from polymeric xylan, acetylated xylose,acetylated glucose, alpha-napthyl acetate or p-nitrophenyl acetate but,typically, not from triacetylglycerol. Such a polypeptide typically doesnot act on acetylated mannan or pectin.

As used herein, a feruloyl esterase (EC 3.1.1.73) is any polypeptidewhich is capable of catalysing a reaction of the form:feruloyl-saccharide+H₂O=ferulate+saccharide. The saccharide may be, forexample, an oligosaccharide or a polysaccharide. It may typicallycatalyse the hydrolysis of the 4-hydroxy-3-methoxycinnamoyl (feruloyl)group from an esterified sugar, which is usually arabinose in ‘natural’substrates. p-nitrophenol acetate and methyl ferulate are typicallypoorer substrates. This enzyme may also be referred to as cinnamoylester hydrolase, ferulic acid esterase or hydroxycinnamoyl esterase. Itmay also be referred to as a hemicellulase accessory enzyme, since itmay help xylanases and pectinases to break down plant cell wallhemicellulose and pectin.

As used herein, a coumaroyl esterase (EC 3.1.1.73) is any polypeptidewhich is capable of catalysing a reaction of the form:coumaroyl-saccharide+H(2)O=coumarate+saccharide. The saccharide may be,for example, an oligosaccharide or a polysaccharide. This enzyme mayalso be referred to as trans-4-coumaroyl esterase, trans-p-coumaroylesterase, p-coumaroyl esterase or p-coumaric acid esterase. This enzymealso falls within EC 3.1.1.73 so may also be referred to as a feruloylesterase.

As used herein, an alpha-galactosidase (EC 3.2.1.22) is any polypeptidewhich is capable of catalysing the hydrolysis of terminal, non-reducingalpha-D-galactose residues in alpha-D-galactosides, including galactoseoligosaccharides, galactomannans, galactans and arabinogalactans. Such apolypeptide may also be capable of hydrolyzing alpha-D-fucosides. Thisenzyme may also be referred to as melibiase.

As used herein, a beta-galactosidase (EC 3.2.1.23) is any polypeptidewhich is capable of catalysing the hydrolysis of terminal non-reducingbeta-D-galactose residues in beta-D-galactosides. Such a polypeptide mayalso be capable of hydrolyzing alpha-L-arabinosides. This enzyme mayalso be referred to as exo-(1->4)-beta-D-galactanase or lactase.

As used herein, a beta-mannanase (EC 3.2.1.78) is any polypeptide whichis capable of catalysing the random hydrolysis of 1,4-beta-D-mannosidiclinkages in mannans, galactomannans and glucomannans. This enzyme mayalso be referred to as mannan endo-1,4-beta-mannosidase orendo-1,4-mannanase.

As used herein, a beta-mannosidase (EC 3.2.1.25) is any polypeptidewhich is capable of catalysing the hydrolysis of terminal, non-reducingbeta-D-mannose residues in beta-D-mannosides. This enzyme may also bereferred to as mannanase or mannase.

A composition as described herein may comprise any pectinase, forexample an endo-polygalacturonase, a pectin methyl esterase, anendo-galactanase, a beta-galactosidase, a pectin acetyl esterase, anendo-pectin lyase, pectate lyase, alpha-rhamnosidase, anexo-galacturonase, an expolygalacturonate lyase, a rhamnogalacturonanhydrolase, a rhamnogalacturonan lyase, a rhamnogalacturonan acetylesterase, a rhamnogalacturonan galacturonohydrolase, axylogalacturonase.

As used herein, an endo-polygalacturonase (EC 3.2.1.15) is anypolypeptide which is capable of catalysing the random hydrolysis of1,4-alpha-D-galactosiduronic linkages in pectate and othergalacturonans. This enzyme may also be referred to as polygalacturonasepectin depolymerase, pectinase, endopolygalacturonase, pectolase, pectinhydrolase, pectin polygalacturonase, poly-alpha-1,4-galacturonideglycanohydrolase, endogalacturonase; endo-D-galacturonase orpoly(1,4-alpha-D-galacturonide) glycanohydrolase.

As used herein, a pectin methyl esterase (EC 3.1.1.11) is any enzymewhich is capable of catalysing the reaction: pectin+n H₂O=nmethanol+pectate. The enzyme may also been known as pectinesterase,pectin demethoxylase, pectin methoxylase, pectin methylesterase,pectase, pectinoesterase or pectin pectylhydrolase.

As used herein, an endo-galactanase (EC 3.2.1.89) is any enzyme capableof catalysing the endohydrolysis of 1,4-beta-D-galactosidic linkages inarabinogalactans. The enzyme may also be known as arabinogalactanendo-1,4-beta-galactosidase, endo-1,4-beta-galactanase, galactanase,arabinogalactanase or arabinogalactan 4-beta-D-galactanohydrolase.

As used herein, a pectin acetyl esterase is defined herein as any enzymewhich has an acetyl esterase activity which catalyses the deacetylationof the acetyl groups at the hydroxyl groups of GalUA residues of pectin.

As used herein, an endo-pectin lyase (EC 4.2.2.10) is any enzyme capableof catalysing the eliminative cleavage of (1→4)-α-D-galacturonan methylester to give oligosaccharides with4-deoxy-6-O-methyl-alpha-D-galact-4-enuronosyl groups at theirnon-reducing ends. The enzyme may also be known as pectin lyase, pectintrans-eliminase; endo-pectin lyase, polymethylgalacturonictranseliminase, pectin methyltranseliminase, pectolyase, PL, PNL or PMGLor (1→4)-6-O-methyl-alpha-D-galacturonan lyase.

As used herein, a pectate lyase (EC 4.2.2.2) is any enzyme capable ofcatalysing the eliminative cleavage of (1→4)-alpha-D-galacturonan togive oligosaccharides with 4-deoxy-alpha-D-galact-4-enuronosyl groups attheir non-reducing ends. The enzyme may also be known polygalacturonictranseliminase, pectic acid transeliminase, polygalacturonate lyase,endopectin methyltranseliminase, pectate transeliminase,endogalacturonate transeliminase, pectic acid lyase, pectic lyase,alpha-1,4-D-endopolygalacturonic acid lyase, PGA lyase, PPase-N,endo-α-1,4-polygalacturonic acid lyase, polygalacturonic acid lyase,pectin trans-eliminase, polygalacturonic acid trans-eliminase or(1→4)-alpha-D-galacturonan lyase.

As used herein, an alpha-rhamnosidase (EC 3.2.1.40) is any polypeptidewhich is capable of catalysing the hydrolysis of terminal non-reducingalpha-L-rhamnose residues in alpha-L-rhamnosides or alternatively inrhamnogalacturonan. This enzyme may also be known asalpha-L-rhamnosidase T, alpha-L-rhamnosidase N or alpha-L-rhamnosiderhamnohydrolase.

As used herein, exo-galacturonase (EC 3.2.1.82) is any polypeptidecapable of hydrolysis of pectic acid from the non-reducing end,releasing digalacturonate. The enzyme may also be known asexo-poly-alpha-galacturonosidase, exo-polygalacturonosidase orexo-polygalacturanosidase.

As used herein, exo-galacturonase (EC 3.2.1.67) is any polypeptidecapable of catalysing:(1,4-alpha-D-galacturonide)_(n)+H₂O=(1,4-alpha-D-galacturonide)_(n-1)+D-galacturonate.The enzyme may also be known as galacturan 1,4-alpha-galacturonidase,exopolygalacturonase, poly(galacturonate) hydrolase,exo-D-galacturonase, exo-D-galacturonanase, exo-poly-D-galacturonase orpoly(1,4-alpha-D-galacturonide) galacturonohydrolase.

As used herein, exopolygalacturonate lyase (EC 4.2.2.9) is anypolypeptide capable of catalysing eliminative cleavage of4-(4-deoxy-alpha-D-galact-4-enuronosyl)-D-galacturonate from thereducing end of pectate, i.e. de-esterified pectin. This enzyme may beknown as pectate disaccharide-lyase, pectate exo-lyase, exopectic acidtranseliminase, exopectate lyase, exopolygalacturonicacid-trans-eliminase, PATE, exo-PATE, exo-PGL or(1→4)-alpha-D-galacturonan reducing-end-disaccharide-lyase.

As used herein, rhamnogalacturonan hydrolase is any polypeptide which iscapable of hydrolyzing the linkage between galactosyluronic acid andrhamnopyranosyl in an endo-fashion in strictly alternatingrhamnogalacturonan structures, consisting of the disaccharide[(1,2)-alpha-L-rhamnoyl-(1,4)-alpha-galactosyluronic acid].

As used herein, rhamnogalacturonan lyase is any polypeptide which is anypolypeptide which is capable of cleavingalpha-L-Rhap-(1→4)-alpha-D-GalpA linkages in an endo-fashion inrhamnogalacturonan by beta-elimination.

As used herein, rhamnogalacturonan acetyl esterase is any polypeptidewhich catalyzes the deacetylation of the backbone of alternatingrhamnose and galacturonic acid residues in rhamnogalacturonan.

As used herein, rhamnogalacturonan galacturonohydrolase is anypolypeptide which is capable of hydrolyzing galacturonic acid from thenon-reducing end of strictly alternating rhamnogalacturonan structuresin an exo-fashion.

As used herein, xylogalacturonase is any polypeptide which acts onxylogalacturonan by cleaving the beta-xylose substituted galacturonicacid backbone in an endo-manner. This enzyme may also be known asxylogalacturonan hydrolase.

As used herein, endo-arabinanase (EC 3.2.1.99) is any polypeptide whichis capable of catalysing endohydrolysis of 1,5-alpha-arabinofuranosidiclinkages in 1,5-arabinans. The enzyme may also be known asendo-arabinase, arabinan endo-1,5-alpha-L-arabinosidase,endo-1,5-alpha-L-arabinanase, endo-alpha-1,5-arabanase; endo-arabanaseor 1,5-alpha-L-arabinan 1,5-alpha-L-arabinanohydrolase.

In addition, one or more (for example two, three, four or all) of anamylase, a protease, a lipase, a ligninase, a hexosyltransferase, aglucuronidase, an expansin, a cellulose induced protein or a celluloseintegrating protein or like protein may be present in a composition asdescribed herein.

“Protease” includes enzymes that hydrolyze peptide bonds (peptidases),as well as enzymes that hydrolyze bonds between peptides and othermoieties, such as sugars (glycopeptidases). Many proteases arecharacterized under EC 3.4 and are suitable for use in the methods asdescribed herein. Some specific types of proteases include, cysteineproteases including pepsin, papain and serine proteases includingchymotrypsins, carboxypeptidases and metalloendopeptidases.

“Lipase” includes enzymes that hydrolyze lipids, fatty acids, andacylglycerides, including phospoglycerides, lipoproteins,diacylglycerols, and the like. In plants, lipids are used as structuralcomponents to limit water loss and pathogen infection. These lipidsinclude waxes derived from fatty acids, as well as cutin and suberin.

“Ligninase” includes enzymes that can hydrolyze or break down thestructure of lignin polymers. Enzymes that can break down lignin includelignin peroxidases, manganese peroxidases, laccases and feruloylesterases, and other enzymes described in the art known to depolymerizeor otherwise break lignin polymers. Also included are enzymes capable ofhydrolyzing bonds formed between hemicellulosic sugars (notablyarabinose) and lignin. Ligninases include but are not limited to thefollowing group of enzymes: lignin peroxidases (EC 1.11.1.14), manganeseperoxidases (EC 1.11.1.13), laccases (EC 1.10.3.2) and feruloylesterases (EC 3.1.1.73).

“Hexosyltransferase” (2.4.1-) includes enzymes which are capable ofcatalysing a transferase reaction, but which can also catalyze ahydrolysis reaction, for example of cellulose and/or cellulosedegradation products. An example of a hexosyltransferase which may beused in the application is a beta-glucanosyltransf erase. Such an enzymemay be able to catalyze degradation of (1,3)(1,4)glucan and/or celluloseand/or a cellulose degradation product. “Glucuronidase” includes enzymesthat catalyze the hydrolysis of a glucoronoside, for exampleβ-glucuronoside to yield an alcohol. Many glucuronidases have beencharacterized and may be suitable for use in the application, forexample beta-glucuronidase (EC 3.2.1.31), hyalurono-glucuronidase (EC3.2.1.36), glucuronosyl-disulfoglucosamine glucuronidase (3.2.1.56),glycyrrhizinate beta-glucuronidase (3.2.1.128) or alpha-D-glucuronidase(EC 3.2.1.139).

A composition as described herein may comprise an expansin orexpansin-like protein, such as a swollenin (see Salheimo et al., Eur. J.Biochem. 269, 4202-4211, 2002) or a swollenin-like protein.

Expansins are implicated in loosening of the cell wall structure duringplant cell growth. Expansins have been proposed to disrupt hydrogenbonding between cellulose and other cell wall polysaccharides withouthaving hydrolytic activity. In this way, they are thought to allow thesliding of cellulose fibers and enlargement of the cell wall. Swollenin,an expansin-like protein contains an N-terminal Carbohydrate BindingModule Family 1 domain (CBD) and a C-terminal expansin-like domain. Forthe purposes of this application, an expansin-like protein orswollenin-like protein may comprise one or both of such domains and/ormay disrupt the structure of cell walls (such as disrupting cellulosestructure), optionally without producing detectable amounts of reducingsugars. A composition as described herein may comprise a celluloseinduced protein, for example the polypeptide product of the cip1 or cip2gene or similar genes (see Foreman et al., J. Biol. Chem. 278(34),31988-31997, 2003), a cellulose/cellulosome integrating protein, forexample the polypeptide product of the cipA or cipC gene, or ascaffoldin or a scaffoldin-like protein. Scaffoldins and celluloseintegrating proteins are multi-functional integrating subunits which mayorganize cellulolytic subunits into a multi-enzyme complex. This isaccomplished by the interaction of two complementary classes of domain,i.e. a cohesion domain on scaffoldin and a dockerin domain on eachenzymatic unit. The scaffoldin subunit also bears a cellulose-bindingmodule (CBM) that mediates attachment of the cellulosome to itssubstrate. A scaffoldin or cellulose integrating protein for thepurposes of this application may comprise one or both of such domains.

A composition as described herein may also comprise a catalase. The term“catalase” means a hydrogen-peroxide: hydrogen-peroxide oxidoreductase(EC 1.11.1.6 or EC 1.11.1.21) that catalyzes the conversion of twohydrogen peroxides to oxygen and two waters. Catalase activity can bedetermined by monitoring the degradation of hydrogen peroxide at 240 nmbased on the following reaction: 2H₂O₂→2H₂O+O₂. The reaction isconducted in 50 mM phosphate pH 7.0 at 25° C. with 10.3 mM substrate(H₂0₂) and approximately 100 units of enzyme per ml. Absorbance ismonitored spectrophotometrically within 16-24 seconds, which shouldcorrespond to an absorbance reduction from 0.45 to 0.4. One catalaseactivity unit can be expressed as one micromole of H₂0₂ degraded perminute at pH 7.0 and 25° C.

The term “amylase” as used herein means enzymes that hydrolyzealpha-1,4-glucosidic linkages in starch, both in amylose andamylopectin, such as alpha-amylase (EC 3.2.1.1), beta-amylase (EC3.2.1.2), glucan 1,4-alpha-glucosidase (EC 3.2.1.3), glucan1,4-alpha-maltotetraohydrolase (EC 3.2.1.60), glucan1,4-alpha-maltohexaosidase (EC 3.2.1.98), glucan1,4-alpha-maltotriohydrolase (EC 3.2.1.116) and glucan1,4-alpha-maltohydrolase (EC 3.2.1.133), and enzymes that hydrolyzealpha-1,6-glucosidic linkages, being the branch-points in amylopectin,such as pullulanase (EC 3.2.1.41) and limit dextinase (EC 3.2.1.142).

A composition as described herein may be composed of a member of each ofthe classes of enzymes mentioned above, several members of one enzymeclass, or any combination of these enzymes classes or helper proteins(i.e. those proteins mentioned herein which do not have enzymaticactivity per se, but do nevertheless assist in lignocellulosicdegradation).

A composition as described herein may be composed of enzymes from (1)commercial suppliers; (2) cloned genes expressing enzymes; (3) broth(such as that resulting from growth of a microbial strain in media,wherein the strains secrete proteins and enzymes into the media; (4)cell lysates of strains grown as in (3); and/or (5) plant materialexpressing enzymes. Different enzymes in a composition as describedherein may be obtained from different sources.

In the uses and methods described herein, the components of thecompositions described above may be provided concomitantly (i.e. as asingle composition per se) or separately or sequentially.

The enzymes can be produced either exogenously in microorganisms,yeasts, fungi, bacteria or plants, then isolated and added, for example,to cellulosic or lignocellulosic material. Alternatively, the enzyme maybe produced in a fermentation that uses (pretreated) lignocellulosicmaterial (such as corn stover or wheat straw) to provide nutrition to anorganism that produces an enzyme(s). In this manner, plants that producethe enzymes may themselves serve as a lignocellulosic material and beadded into lignocellulosic material.

In an embodiment the composition is a whole fermentation broth. In anembodiment the composition is in the form of a whole fermentation brothof a fungus, preferably Rasamsonia. The whole fermentation broth can beprepared from fermentation of non-recombinant and/or recombinantfilamentous fungi. In an embodiment the filamentous fungus is arecombinant filamentous fungus comprising one or more genes which can behomologous or heterologous to the filamentous fungus. In an embodiment,the filamentous fungus is a recombinant filamentous fungus comprisingone or more genes which can be homologous or heterologous to thefilamentous fungus wherein the one or more genes encode enzymes that candegrade a cellulosic substrate. The whole fermentation broth maycomprise any of the polypeptides described above or any combinationthereof.

Preferably, the composition is a whole fermentation broth wherein thecells are killed. The whole fermentation broth may contain organicacid(s) (used for killing the cells), killed cells and/or cell debris,and culture medium.

Generally, the filamentous fungi is cultivated in a cell culture mediumsuitable for production of enzymes capable of hydrolyzing a cellulosicsubstrate. The cultivation takes place in a suitable nutrient mediumcomprising carbon and nitrogen sources and inorganic salts, usingprocedures known in the art. Suitable culture media, temperature rangesand other conditions suitable for growth and cellulase and/orhemicellulase and/or pectinase production are known in the art. Thewhole fermentation broth can be prepared by growing the filamentousfungi to stationary phase and maintaining the filamentous fungi underlimiting carbon conditions for a period of time sufficient to express avariant polypeptide as described herein and/or one or more cellulasesand/or hemicellulases and/or pectinases. Once enzymes, such as thevariant polypeptide as described herein and/or cellulases and/orhemicellulases and/or pectinases, are secreted by the filamentous fungiinto the fermentation medium, the whole fermentation broth can be used.The whole fermentation broth as described herein may comprisefilamentous fungi. In some embodiments, the whole fermentation brothcomprises the unfractionated contents of the fermentation materialsderived at the end of the fermentation. Typically, the wholefermentation broth comprises the spent culture medium and cell debrispresent after the filamentous fungi is grown to saturation, incubatedunder carbon-limiting conditions to allow protein synthesis(particularly, expression of cellulases and/or hemicellulases and/orpectinases). In some embodiments, the whole fermentation broth comprisesthe spent cell culture medium, extracellular enzymes and filamentousfungi. In some embodiments, the filamentous fungi present in wholefermentation broth can be lysed, permeabilized, or killed using methodsknown in the art to produce a cell-killed whole fermentation broth. Inan embodiment, the whole fermentation broth is a cell-killed wholefermentation broth, wherein the whole fermentation broth containing thefilamentous fungi cells are lysed or killed. In some embodiments, thecells are killed by lysing the filamentous fungi by chemical and/or pHtreatment to generate the cell-killed whole broth of a fermentation ofthe filamentous fungi. In some embodiments, the cells are killed bylysing the filamentous fungi by chemical and/or pH treatment andadjusting the pH of the cell-killed fermentation mix to a suitable pH.In an embodiment, the whole fermentation broth comprises a first organicacid component comprising at least one 1-5 carbon organic acid and/or asalt thereof and a second organic acid component comprising at least 6or more carbon organic acid and/or a salt thereof. In an embodiment, thefirst organic acid component is acetic acid, formic acid, propionicacid, a salt thereof, or any combination thereof and the second organicacid component is benzoic acid, cyclohexanecarboxylic acid,4-methylvaleric acid, phenylacetic acid, a salt thereof, or anycombination thereof.

The term “whole fermentation broth” as used herein refers to apreparation produced by cellular fermentation that undergoes no orminimal recovery and/or purification. For example, whole fermentationbroths are produced when microbial cultures are grown to saturation,incubated under carbon-limiting conditions to allow protein synthesis(e.g., expression of enzymes by host cells) and secretion into cellculture medium. Typically, the whole fermentation broth isunfractionated and comprises spent cell culture medium, extracellularenzymes, and microbial, preferably non-viable, cells.

If needed, the whole fermentation broth can be fractionated and the oneor more of the fractionated contents can be used. For instance, thekilled cells and/or cell debris can be removed from a whole fermentationbroth to provide a composition that is free of these components.

The whole fermentation broth may further comprise a preservative and/oranti-microbial agent. Such preservatives and/or agents are known in theart.

The whole fermentation broth as described herein is typically a liquid,but may contain insoluble components, such as killed cells, cell debris,culture media components, and/or insoluble enzyme(s). In someembodiments, insoluble components may be removed to provide a clarifiedwhole fermentation broth.

In an embodiment, the whole fermentation broth may be supplemented withone or more enzyme activities that are not expressed endogenously, orexpressed at relatively low level by the filamentous fungi, to improvethe degradation of the cellulosic substrate, for example, to fermentablesugars such as glucose or xylose. The supplemental enzyme(s) can beadded as a supplement to the whole fermentation broth and the enzymesmay be a component of a separate whole fermentation broth, or may bepurified, or minimally recovered and/or purified.

In an embodiment, the whole fermentation broth comprises a wholefermentation broth of a fermentation of a recombinant filamentous fungioverexpressing one or more enzymes to improve the degradation of thecellulosic substrate. Alternatively, the whole fermentation broth cancomprise a mixture of a whole fermentation broth of a fermentation of anon-recombinant filamentous fungus and a recombinant filamentous fungusoverexpressing one or more enzymes to improve the degradation of thecellulosic substrate. In an embodiment, the whole fermentation brothcomprises a whole fermentation broth of a fermentation of a filamentousfungi overexpressing a variant polypeptide as described herein. In anembodiment, the whole fermentation broth comprises a whole fermentationbroth of a fermentation of a filamentous fungi overexpressing anotherbeta-glucosidase. Alternatively, the whole fermentation broth for use inthe present methods and compositions can comprise a mixture of a wholefermentation broth of a fermentation of a non-recombinant filamentousfungus expressing a beta-glucosidase and a whole fermentation broth of afermentation of a recombinant filamentous fungi overexpressing a variantpolypeptide as described herein. Alternatively, the whole fermentationbroth for use in the present methods and compositions can comprise amixture of a whole fermentation broth of a fermentation of a recombinantfilamentous fungus not expressing any beta-glucosidase and a wholefermentation broth of a fermentation of a recombinant filamentous fungioverexpressing a variant polypeptide as described herein.

Use of the Polypeptides and Compositions

The polypeptides and compositions as described herein may be used inmany different applications. For instance, they may be used to producefermentable sugars. The fermentable sugars can then, as part of abiofuel method, be converted into biogas or ethanol, butanol,isobutanol, 2-butanol or other suitable substances. So, by fermentablesugars is meant sugars which can be consumed by a microorganism orconverted by a microorganism in another product. Alternatively, thevariant polypeptide as described herein may be used as enzyme, forinstance in production of food products, in detergent compositions, inthe paper and pulp industry and in antibacterial formulations, inpharmaceutical products such as throat lozenges, toothpastes, andmouthwash. Some of the uses will be illustrated in more detail below.

In the uses and methods described below, the components of thecompositions described above may be provided concomitantly (i.e. as asingle composition per se) or separately or sequentially.

The application also relates to the use of the variant polypeptide asdescribed herein and compositions in industrial methods.

In principle, a polypeptide or composition as described herein may beused in any method which requires the treatment of a material whichcomprises polysaccharide. Thus, a polypeptide and/or composition asdescribed herein may be used in the treatment of polysaccharidematerial. Herein, polysaccharide material is a material which comprisesor consists essential of one or, more typically, more than onepolysaccharide.

Typically, plants and material derived therefrom comprise significantquantities of non-starch polysaccharide material. Accordingly, a variantpolypeptide as described herein may be used in the treatment of a plantor fungal material or a material derived therefrom.

Cellulosic Material

“Substrate” is used to refer to a substance that comprises carbohydratematerial, which may be treated with polypeptides as described herein, sothat the carbohydrate material is modified. The substrate may bepretreated or non-pretreated substrate. In addition to the carbohydratematerial, the substrate may contain any other component including, butnot limited to, non-carbohydrate material and starch. Substrate may becellulosic material. Substrate may also be lignocellulosic material.

Cellulosic and lignocellulosic materials are abundant in nature and havegreat value as alternative energy source. Second generation biofuels,also known as advanced biofuels, are fuels that can be manufactured fromvarious types of these materials. The materials can be derived fromplants, but can also include animal materials. The composition of thematerials varies. The major component is cellulose (in general 35-50%),followed by xylan (a type of hemicellulose, in general 20-35%). Somematerials also comprise lignin (in general 10-25%). The materials alsomay comprise minor components such as proteins, oils and ash (orinorganic compounds).

The materials contain a variety of carbohydrates. The term carbohydrateis most common in biochemistry, where it is a synonym of saccharide.Carbohydrates are divided into four chemical groupings: monosaccharides,disaccharides, oligosaccharides, and polysaccharides. In general,monosaccharides and disaccharides, which are smaller (lower molecularweight) carbohydrates, are commonly referred to as sugars. The enzymaticconversion (such as hydrolysis) of polysaccharides to soluble sugars,for example glucose, gluconic acid, xylose, arabinose, galactose,fructose, mannose, rhamnose, ribose, D-galacturonic acid and otherhexoses and pentoses occurs under the action of different enzymes actingin concert.

A composition as described herein may be tailored in view of theparticular substrate which is to be used. That is to say, the spectrumof activities in a composition as described herein may vary depending onthe substrate in question.

The enzymes used to hydrolyze the substrate can be produced eitherexogenously in microorganisms such as yeasts, fungi, bacteria or plants,then isolated and added to the substrate. Alternatively, the enzymes canbe produced, but not isolated, and a whole fermentation broth can beadded to the substrate. Alternatively, the whole fermentation broth maybe treated to prevent further microbial growth (for example, by heatingor addition of antimicrobial agents), then added to the substrate. Thewhole fermentation broth may include the organism producing theenzyme(s). Alternatively, the enzyme may be produced in a fermentationthat uses substrate to provide nutrition to an organism that producesthe enzymes. In this manner, plants that produce the enzymes may serveas the substrate and be added to substrate.

Example of suitable cellulosic and/or lignocellulosic materials include,but are not limited to, virgin biomass and/or non-virgin biomass such asagricultural biomass, herbaceous material, agricultural residues,forestry residues, commercial organics, construction and demolitiondebris, municipal solid waste, waste paper, yard waste, and pulp andpaper mill residues. Common forms of biomass include trees, shrubs andgrasses, wheat, wheat straw, sugar cane bagasse, corn, corn husks, corncobs, corn kernel including fiber from kernels, products and by-productsfrom milling of grains such as corn, wheat and barley (including wetmilling and dry milling) often called “bran or fiber” as well asmunicipal solid waste, waste paper and yard waste. “Agriculturalbiomass” includes branches, bushes, canes, corn and corn husks, energycrops, forests, fruits, flowers, grains, grasses, herbaceous crops,leaves, bark, needles, logs, roots, saplings, short rotation woodycrops, shrubs, switch grasses, trees, vegetables, fruit peels, vines,sugar beet pulp, wheat middlings, oat hulls, and hard and soft woods(not including woods with deleterious materials). In addition,agricultural biomass includes organic waste materials generated fromagricultural methods including farming and forestry activities,specifically including forestry wood waste. Agricultural biomass may beany of the aforestated singularly or in any combination or mixturethereof. Further examples of suitable biomass are orchard primings,chaparral, mill waste, urban wood waste, municipal waste, logging waste,forest thinnings, short-rotation woody crops, industrial waste, wheatstraw, oat straw, rice straw, cane straw, barley straw, rye straw, flaxstraw, soy hulls, rice hulls, rice straw, corn gluten feed, oat hulls,sugar cane, corn stover, corn stalks, corn cobs, corn fiber, corn husks,prairie grass, gamagrass, foxtail; sugar beet pulp, citrus fruit pulp,seed hulls, cellulosic animal wastes, lawn clippings, cotton, seaweed,trees, shrubs, grasses, wheat, wheat straw, sugar cane bagasse, corn,corn husks, corn hobs, corn kernel, fiber from kernels, products andby-products from wet or dry milling of grains, municipal solid waste,waste paper, yard waste, herbaceous material, agricultural residues,forestry residues, municipal solid waste, waste paper, pulp, paper millresidues, branches, bushes, canes, corn, corn husks, an energy crop,forest, a fruit, a flower, a grain, a grass, a herbaceous crop, a leaf,bark, a needle, a log, a root, a sapling, a shrub, switch grass, a tree,a vegetable, fruit peel, a vine, sugar beet pulp, wheat middlings, oathulls, hard or soft wood, organic waste material generated from anagricultural method, forestry wood waste, or a combination of any two ormore thereof.

Apart from virgin biomass or feedstocks already methoded in food andfeed or paper and pulping industries, the biomass/feedstock mayadditionally be pretreated with heat, mechanical and/or chemicalmodification or any combination of such methods in order to enhanceenzymatic degradation.

Pretreatment

Before enzymatic treatment, the feedstock may optionally be pretreatedwith heat, mechanical and/or chemical modification or any combination ofsuch methods in order to to enhance the accessibility of the substrateto enzymatic hydrolysis and/or hydrolyse the hemicellulose and/orsolubilize the hemicellulose and/or cellulose and/or lignin, in any wayknown in the art. The pretreatment may comprise exposing thelignocellulosic material to (hot) water, steam (steam explosion), anacid, a base, a solvent, heat, a peroxide, ozone, mechanical shredding,grinding, milling or rapid depressurization, or a combination of any twoor more thereof. This chemical pretreatment is often combined withheat-pretreatment, e.g. between 150 and 220° C. for 1 to 30 minutes.

In an embodiment the lignocellulosic material is pretreated beforeand/or during the enzymatic hydrolysis. Pretreatment methods are knownin the art and include, but are not limited to, heat, mechanical,chemical modification, biological modification and any combinationthereof. Pretreatment is typically performed in order to enhance theaccessibility of the lignocellulosic material to enzymatic hydrolysisand/or hydrolyse the hemicellulose and/or solubilize the hemicelluloseand/or cellulose and/or lignin, in the lignocellulosic material. In anembodiment, the pretreatment comprises treating the lignocellulosicmaterial with steam explosion, hot water treatment or treatment withdilute acid or dilute base. Examples of pretreatment methods include,but are not limited to, steam treatment (e.g. treatment at 100-260° C.,at a pressure of 7-45 bar, at neutral pH, for 1-10 minutes), dilute acidtreatment (e.g. treatment with 0.1-5% H₂SO₄ and/or SO₂ and/or HNO₃and/or HCl, in presence or absence of steam, at 120-200° C., at apressure of 2-15 bar, at acidic pH, for 2-30 minutes), organosolvtreatment (e.g. treatment with 1-1.5% H₂SO₄ in presence of organicsolvent and steam, at 160-200° C., at a pressure of 7-30 bar, at acidicpH, for 30-60 minutes), lime treatment (e.g. treatment with 0.1-2%NaOH/Ca(OH)₂ in the presence of water/steam at 60-160° C., at a pressureof 1-10 bar, at alkaline pH, for 60-4800 minutes), ARP treatment (e.g.treatment with 5-15% NH₃, at 150-180° C., at a pressure of 9-17 bar, atalkaline pH, for 10-90 minutes), AFEX treatment (e.g. treatmentwith >15% NH₃, at 60-140° C., at a pressure of 8-20 bar, at alkaline pH,for 5-30 minutes).

Hydrolysis

The application also relates to a method for degrading a substrate, themethod comprising the step of contacting the substrate with a variantpolypeptide as described herein and/or a composition as describedherein. The degradation may result in the production of a sugar.

The application also relates to a method for the treatment of asubstrate which method comprises the step of contacting the substratewith a variant polypeptide as described herein and/or a composition asdescribed herein. The treatment may result in the production of a sugar.

The application also relates to a method of producing a sugar from asubstrate, comprising the steps of (a) enzymatic hydrolysis of thesubstrate using a variant polypeptide as described herein and/or acomposition as described herein to obtain enzymatically hydrolysedsubstrate, and (b), optionally, recovery of the enzymatically hydrolysedsubstrate. The enzymatically hydrolysed substrate may comprise a sugar.

The application also relates to a process for the preparation of sugarfrom lignocellulosic material comprising the steps of (a) hydrolysingthe lignocellulosic material with a variant polypeptide as describedherein and/or an enzyme composition as described herein to obtain thesugar, and optionally, recovering the sugar.

In an embodiment the substrate comprises a carbohydrate material. In anembodiment the substrate comprises cellulose and/or hemicellulose. In anembodiment the substrate comprises a cellulosic material. In anembodiment the substrate comprises a lignocellulosic material.

In an embodiment the pH of the above methods is between 3.0 and 6.5,preferably between 3.5 and 5.5, more preferably between 4.0 and 5.0.

After the methods have been performed, the resulting material may besubjected to at least one solid/liquid separation. The methods andconditions of solid/liquid separation will depend on the type ofsubstrate used and are well within the scope of the skilled artisan.Examples include, but are not limited to, centrifugation, cyclonicseparation, filtration, decantation, sieving and sedimentation. In apreferred embodiment the solid/liquid separation is performed bycentrifugation or sedimentation. During solid/liquid separation, meansand/or aids for improving the separation may be used.

In an embodiment the substrate is subjected to a pretreatment stepbefore the above methods. In an embodiment the substrate is subjected toa washing step before the above methods. In an embodiment the substrateis subjected to at least one solid/liquid separation before the abovemethods. So, before subjecting the substrate to any of the abovemethods, it can be subjected to at least one solid/liquid separation.The solid/liquid separation may be done before and/or after thepretreatment step. Suitable methods and conditions for a solid/liquidseparation have been described above.

In an embodiment the application relates to methods wherein theenzymatically hydrolysed substrate is subjected to a solid/liquidseparation step followed by a detoxification step and/or a concentrationstep.

In the methods as described herein substrate may be added to the one ormore containers. In an embodiment the variant polypeptide as describedherein and/or composition as described herein is already present in theone or more containers before the substrate is added. In anotherembodiment the variant polypeptide as described herein and/orcomposition as described herein may be added to the one or morecontainers. In an embodiment the substrate is already present in the oneor more containers before the variant polypeptide as described hereinand/or the composition as described herein is added. In an embodimentboth the substrate and the variant polypeptide as described hereinand/or the composition as described herein are added simultaneously tothe one or more containers. The composition present in the one or morecontainers may be an aqueous composition.

The above methods may comprise a liquefaction step wherein the substrateis liquefied, and a saccharification step wherein the liquefiedsubstrate is saccharified. The liquefaction step is sometimes calledpresaccharification step. In an embodiment the methods comprise at leasta liquefaction step wherein the substrate is liquefied in at least afirst container, and a saccharification step wherein the liquefiedsubstrate is saccharified in the at least first container and/or in atleast a second container. Saccharification can be done in the samecontainer as the liquefaction (i.e. the at least first container), itcan also be done in a separate container (i.e. the at least secondcontainer). So, in the methods as described herein liquefaction andsaccharification may be combined. Alternatively, the liquefaction andsaccharification may be separate steps. Liquefaction andsaccharification may be performed at different temperatures, but mayalso be performed at a single temperature. In an embodiment thetemperature of the liquefaction is higher than the temperature of thesaccharification. Liquefaction is preferably carried out at atemperature of 60-75° C. and saccharification is preferably carried outat a temperature of 50-65° C.

The methods can be performed in one or more containers, but can also beperformed in one or more tubes or any other continuous system. This alsoholds true when the above methods comprises a liquefaction step and asaccharification step. The liquefaction step can be performed in one ormore containers, but can also be performed in one or more tubes or anyother continuous system and/or the saccharification step can beperformed in one or more containers, but can also be performed in one ormore tubes or any other continuous system. Examples of containers to beused in the present application include, but are not limited to,fed-batch stirred containers, batch stirred containers, continuous flowstirred containers with ultrafiltration, and continuous plug-flow columnreactors. Stirring can be done by one or more impellers, pumps and/orstatic mixers.

The polypeptides and/or compositions used in the above methods may beadded before and/or during the methods. As indicated above, when thesubstrate is subjected to a solid/liquid separation before the abovemethods, the polypeptides and/or compositions used in the above methodsmay be added before the solid/liquid separation. Alternatively, they mayalso be added after solid/liquid separation or before and aftersolid/liquid separation. The polypeptides and/or compositions may alsobe added during the above methods. In case the above methods comprise aliquefaction step and saccharification step, additional polypeptidesand/or compositions may be added during and/or after the liquefactionstep. The additional polypeptides and/or compositions may be addedbefore and/or during the saccharification step. Additional polypeptidesand/or compositions may also be added after the saccharification step.

In an embodiment the total method time is 10 hours or more, 12 hours ormore, 14 hours or more, 16 hours or more, 18 hours or more, 20 hours ormore, 30 hours or more, 40 hours or more, 50 hours or more, 60 hours ormore, 70 hours or more, 80 hours or more, 90 hours or more, 100 hours ormore, 110 hours or more, 120 hours or more, 130 hours or more, 140 hoursor more, 150 hours or more, 160 hours or more, 170 hours or more, 180hours or more, 190 hours or more, 200 hours or more.

In an embodiment, the total method time is 10 to 300 hours, 16 to 275hours, preferably 20 to 250 hours, more preferably 30 to 200 hours, mostpreferably 40 to 150 hours.

The viscosity of the substrate in the one or more containers used forthe above methods is kept between 10 and 4000 cP, between 10 and 2000cP, preferably between 10 and 1000 cP.

Incubation of substrate under the above conditions results in release orliberation of a substantial amount of sugars from the substrate. Bysubstantial amount is meant at least 20%, 30%, 40%, 50%, 60%, 70%, 80%,90%, 95% or more of the available sugars.

In case the methods comprises a liquefaction step and a saccharificationstep, the viscosity of the substrate in the liquefaction step is keptbetween 10 and 4000 cP, between 10 and 2000 cP, preferably between 10and 1000 cP and/or the viscosity of the substrate in thesaccharification step is kept between 10 and 1000 cP, between 10 and 900cP, preferably between 10 and 800 cP.

The viscosity can be determined with a Brookfield DV III Rheometer atthe temperature used for the above methods.

Significantly, the above methods may be carried out using high levels ofdry matter (of the substrate). In an embodiment the dry matter contentat the end of the above methods is 5 wt % or higher, 6 wt % or higher, 7wt % or higher, 8 wt % or higher, 9 wt % or higher, 10 wt % or higher,11 wt % or higher, 12 wt % or higher, 13 wt % or higher, 14 wt % orhigher, 15 wt % or higher, 16 wt % or higher, 17 wt % or higher, 18 wt %or higher, 19 wt % or higher, 20 wt % or higher, 21 wt % or higher, 22wt % or higher, 23 wt % or higher, 24 wt % or higher, 25 wt % or higher,26 wt % or higher, 27 wt % or higher, 28 wt % or higher, 29 wt % orhigher, 30 wt % or higher, 31 wt % or higher, 32 wt % or higher, 33 wt %or higher, 34 wt % or higher, 35 wt % or higher, 36 wt % or higher, 37wt % or higher, 38 wt % or higher or 39 wt % or higher. In an embodimentthe dry matter content at the end of the above methods is between 5 wt%-40 wt %, 6 wt %-40 wt %, 7 wt %-40 wt %, 8 wt %-40 wt %, 9 wt %-40 wt%, 10 wt %-40 wt %, 11 wt %-40 wt %, 12 wt %-40 wt %, 13 wt %-40 wt %,14 wt %-40 wt %, 15 wt %-40 wt %, 16 wt %-40 wt %, 17 wt %-40 wt %, 18wt %-40 wt %, 19 wt %-40 wt %, 20 wt %-40 wt %, 21 wt %-40 wt %, 22 wt%-40 wt %, 23 wt %-40 wt %, 24 wt %-40 wt %, 25 wt %-40 wt %, 26 wt %-40wt %, 27 wt %-40 wt %, 28 wt %-40 wt %, 29 wt %-40 wt %, 30 wt %-40 wt%, 31 wt %-40 wt %, 32 wt %-40 wt %, 33 wt %-40 wt %, 34 wt %-40 wt %,35 wt %-40 wt %, 36 wt %-40 wt %, 37 wt %-40 wt %, 38 wt %-40 wt %, 39wt %-40 wt %.

In an embodiment the dry matter content at the end of the liquefactionstep of the above methods is 5 wt % or higher, 6 wt % or higher, 7 wt %or higher, 8 wt % or higher, 9 wt % or higher, 10 wt % or higher, 11 wt% or higher, 12 wt % or higher, 13 wt % or higher, 14 wt % or higher, 15wt % or higher, 16 wt % or higher, 17 wt % or higher, 18 wt % or higher,19 wt % or higher, 20 wt % or higher, 21 wt % or higher, 22 wt % orhigher, 23 wt % or higher, 24 wt % or higher, 25 wt % or higher, 26 wt %or higher, 27 wt % or higher, 28 wt % or higher, 29 wt % or higher, 30wt % or higher, 31 wt % or higher, 32 wt % or higher, 33 wt % or higher,34 wt % or higher, 35 wt % or higher, 36 wt % or higher, 37 wt % orhigher, 38 wt % or higher or 39 wt % or higher. In an embodiment the drymatter content at the end of the liquefaction step of the the abovemethods is between 5 wt %-40 wt %, 6 wt %-40 wt %, 7 wt %-40 wt %, 8 wt%-40 wt %, 9 wt %-40 wt %, 10 wt %-40 wt %, 11 wt %-40 wt %, 12 wt %-40wt %, 13 wt %-40 wt %, 14 wt %-40 wt %, 15 wt %-40 wt %, 16 wt %-40 wt%, 17 wt %-40 wt %, 18 wt %-40 wt %, 19 wt %-40 wt %, 20 wt %-40 wt %,21 wt %-40 wt %, 22 wt %-40 wt %, 23 wt %-40 wt %, 24 wt %-40 wt %, 25wt %-40 wt %, 26 wt %-40 wt %, 27 wt %-40 wt %, 28 wt %-40 wt %, 29 wt%-40 wt %, 30 wt %-40 wt %, 31 wt %-40 wt %, 32 wt %-40 wt %, 33 wt %-40wt %, 34 wt %-40 wt %, 35 wt %-40 wt %, 36 wt %-40 wt %, 37 wt %-40 wt%, 38 wt %-40 wt %, 39 wt %-40 wt %.

In an embodiment the dry matter content at the end of thesaccharification step of the above methods is 5 wt % or higher, 6 wt %or higher, 7 wt % or higher, 8 wt % or higher, 9 wt % or higher, 10 wt %or higher, 11 wt % or higher, 12 wt % or higher, 13 wt % or higher, 14wt % or higher, 15 wt % or higher, 16 wt % or higher, 17 wt % or higher,18 wt % or higher, 19 wt % or higher, 20 wt % or higher, 21 wt % orhigher, 22 wt % or higher, 23 wt % or higher, 24 wt % or higher, 25 wt %or higher, 26 wt % or higher, 27 wt % or higher, 28 wt % or higher, 29wt % or higher, 30 wt % or higher, 31 wt % or higher, 32 wt % or higher,33 wt % or higher, 34 wt % or higher, 35 wt % or higher, 36 wt % orhigher, 37 wt % or higher, 38 wt % or higher or 39 wt % or higher. In anembodiment the dry matter content at the end of the saccharificationstep of the above methods is between 5 wt %-40 wt %, 6 wt %-40 wt %, 7wt %-40 wt %, 8 wt %-40 wt %, 9 wt %-40 wt %, 10 wt %-40 wt %, 11 wt%-40 wt %, 12 wt %-40 wt %, 13 wt %-40 wt %, 14 wt %-40 wt %, 15 wt %-40wt %, 16 wt %-40 wt %, 17 wt %-40 wt %, 18 wt %-40 wt %, 19 wt %-40 wt%, 20 wt %-40 wt %, 21 wt %-40 wt %, 22 wt %-40 wt %, 23 wt %-40 wt %,24 wt %-40 wt %, 25 wt %-40 wt %, 26 wt %-40 wt %, 27 wt %-40 wt %, 28wt %-40 wt %, 29 wt %-40 wt %, 30 wt %-40 wt %, 31 wt %-40 wt %, 32 wt%-40 wt %, 33 wt %-40 wt %, 34 wt %-40 wt %, 35 wt %-40 wt %, 36 wt %-40wt %, 37 wt %-40 wt %, 38 wt %-40 wt %, 39 wt %-40 wt %.

In an embodiment oxygen is added during the above methods. In anembodiment oxygen is added during at least a part of the above methods.Oxygen can be added continuously or discontinuously during the abovemethods. In an embodiment oxygen is added one or more times during theabove methods. In an embodiment oxygen may be added before the abovemethods, during the addition of cellulosic material to a container usedfor the above methods, during the addition of enzyme to a container usedfor the above methods, during a part of the above methods, during thewhole methods or any combination thereof. Oxygen is added to the one ormore containers used in the above methods.

Oxygen can be added in several forms. For example, oxygen can be addedas oxygen gas, oxygen-enriched gas, such as oxygen-enriched air, or air.Oxygen may also be added by means of in situ oxygen generation. Forexample, oxygen may be generated by electrolysis, oxygen may be producedenzymatically, e.g. by the addition of peroxide, or oxygen may beproduced chemically, e.g. by an oxygen generating system such as KHSO₅.For example, oxygen is produced from peroxide by catalase. The peroxidecan be added in the form of dissolved peroxide or generated by anenzymatic or chemical reaction. In case catalase is used as enzyme toproduce oxygen, catalase present in the enzyme composition for thehydrolysis can be used or catalase can be added for this purpose.

Examples how to add oxygen include, but are not limited to, addition ofoxygen by means of sparging, electrolysis, chemical addition of oxygen,filling the one or more containers used in the the above methods fromthe top (plunging the hydrolysate into the tank and consequentlyintroducing oxygen into the hydrolysate) and addition of oxygen to theheadspace of said one or more containers. When oxygen is added to theheadspace of the container(s), sufficient oxygen necessary for thehydrolysis reaction may be supplied. In general, the amount of oxygenadded to the container(s) can be controlled and/or varied. Restrictionof the oxygen supplied is possible by adding only oxygen during part ofthe hydrolysis time in said container(s). Another option is addingoxygen at a low concentration, for example by using a mixture of air andrecycled air (air leaving the container) or by “diluting” air with aninert gas. Increasing the amount of oxygen added can be achieved byaddition of oxygen during longer periods of the hydrolysis time, byadding the oxygen at a higher concentration or by adding more air.Another way to control the oxygen concentration is to add an oxygenconsumer and/or an oxygen generator. Oxygen can be introduced, forexample blown, into the liquid hydrolysis container contents ofsubstrate. It can also be blown into the headspace of the container.

In an embodiment oxygen is added to the one or more containers used inthe above methods before and/or during and/or after the addition of thesubstrate to said one or more containers. The oxygen may be introducedtogether with the substrate that enters the hydrolysis container(s). Theoxygen may be introduced into the material stream that will enter thecontainer(s) or with part of the container(s) contents that passes anexternal loop of the container(s).

In an embodiment the container(s) used in the the above methods and/orthe polypeptide production methods have a volume of at least 1 m³.Preferably, the containers have a volume of at least 1 m³, at least 2m³, at least 3 m³, at least 4 m³, at least 5 m³, at least 6 m³, at least7 m³, at least 8 m³, at least 9 m³, at least 10 m³, at least 15 m³, atleast 20 m³, at least 25 m³, at least 30 m³, at least 35 m³, at least 40m³, at least 45 m³, at least 50 m³, at least 60 m³, at least 70 m³, atleast 75 m³, at least 80 m³, at least 90 m³, at least 100 m³, at least200 m³, at least 300 m³, at least 400 m³, at least 500 m³, at least 600m³, at least 700 m³, at least 800 m³, at least 900 m³, at least 1000 m³,at least 1500 m³, at least 2000 m³, at least 2500 m³. In general, thecontainer(s) will be smaller than 3000 m³ or 5000 m³. In case severalcontainers are used in the above methods, they may have the same volume,but also may have a different volume. In case the above methodscomprises a separate liquefaction step and saccharification step thecontainer(s) used for the liquefaction step and the container(s) usedfor the saccharification step may have the same volume, but also mayhave a different volume.

Hydrolysis and fermentation (see below), separate or simultaneous,include, but are not limited to, separate hydrolysis and fermentation(SHF); simultaneous saccharification and fermentation (SSF);simultaneous saccharification and co-fermentation (SSCF); hybridhydrolysis and fermentation (HHF); separate hydrolysis andco-fermentation (SHCF); hybrid hydrolysis and co-fermentation (HHCF);and direct microbial conversion (DMC), also sometimes calledconsolidated biomethoding (CBP).

Fermentation

The present application also relates to a method of producing afermentation product, the method comprising the steps of (a) treating asubstrate by contacting the substrate with a variant polypeptide asdescribed herein and/or a composition as described herein, and (b)fermenting the resulting material to produce the fermentation product.The resulting material may comprise a sugar.

The present application also relates to a method of producing afermentation product, the method comprising the steps of (a)enzymatically hydrolysing a substrate with a variant polypeptide asdescribed herein and/or a composition as described herein, (b)fermenting the enzymatically hydrolysed cellulosic substrate to producea fermentation product, and (c) optionally, recovering the fermentationproduct.

The present application also relates to a process for producing afermentation product from a lignocellulosic material, which processcomprises the steps of (a) hydrolysing the lignocellulosic material witha variant polypeptide as described herein and/or a composition asdescribed herein to obtain a sugar, (b) fermenting the obtained sugar bycontacting the obtained sugar with a fermenting microorganism to producethe fermentation product, and (c) optionally, recovering thefermentation product.

For instance, in the method as described herein a variant polypeptide asdescribed herein and/or a composition as described herein acts on asubstrate, so as to convert this substrate to sugars andoligosaccharides for the production of fermentation products.

The application thus also provides a method of producing a fermentationproduct, which method comprises (a) degrading a substrate using a methodas described herein, and (b) fermentation of the resulting material,thereby to prepare a fermentation product. The above methods ofproducing a fermentation product may optionally comprise recovery of thefermentation product.

In an embodiment the fermentation (i.e. step b) is performed in one ormore containers. In an embodiment the fermentation is done by an alcoholproducing microorganism to produce alcohol. In an embodiment thefermentation is done by an organic acid producing microorganism toproduce an organic acid. The fermentation by an alcohol producingmicroorganism to produce alcohol can be done in the same container(s)wherein the step (a) is performed. Alternatively, the fermentation by analcohol producing microorganism to produce alcohol and the fermentationby an organic acid producing microorganism to produce an organic acidcan be performed in one or more separate containers, but may also bedone in one or more of the same containers.

In an embodiment the fermentation is done by a yeast. In an embodimentthe alcohol producing microorganism and/or the organic acid producingmicroorganism is a yeast. In an embodiment the alcohol producingmicroorganism is able to ferment at least a C5 sugar and at least a C6sugar. In an embodiment the organic acid producing microorganism is ableto ferment at least a C6 sugar. In an embodiment the alcohol producingmicroorganism and the organic acid producing microorganism are differentmicroorganisms. In another embodiment the alcohol producingmicroorganism and the organic acid producing microorganism are the samemicroorganism, i.e. the alcohol producing microorganism is also able toproduce organic acid such as succinic acid.

In a further aspect, the application thus includes fermentation methodsin which a microorganism is used for the fermentation of a carbon sourcecomprising sugar(s), e.g. glucose, L-arabinose and/or xylose. The carbonsource may include any carbohydrate oligomer or polymer comprisingL-arabinose, xylose or glucose units, such as e.g. lignocellulose,xylans, cellulose, starch, arabinan and the like. For release of xyloseor glucose units from such carbohydrates, appropriate carbohydrases(such as xylanases, glucanases, amylases and the like) may be added tothe fermentation medium or may be produced by the modified host cell. Inthe latter case, the modified host cell may be genetically engineered toproduce and excrete such carbohydrases. An additional advantage of usingoligo- or polymeric sources of glucose is that it enables to maintain alow(er) concentration of free glucose during the fermentation, e.g. byusing rate-limiting amounts of the carbohydrases. This, in turn, willprevent repression of systems required for metabolism and transport ofnon-glucose sugars such as xylose. In a preferred method the modifiedhost cell ferments both the L-arabinose (optionally xylose) and glucose,preferably simultaneously in which case preferably a modified host cellis used which is insensitive to glucose repression to prevent diauxicgrowth. In addition to a source of L-arabinose, optionally xylose (andglucose) as carbon source, the fermentation medium will further comprisethe appropriate ingredient required for growth of the modified hostcell. Compositions of fermentation media for growth of microorganismssuch as yeasts or filamentous fungi are well known in the art.

The fermentation time may be shorter than in conventional fermentationat the same conditions, wherein part of the enzymatic hydrolysis stillhas to take part during fermentation. In one embodiment, thefermentation time is 100 hours or less, 90 hours or less, 80 hours orless, 70 hours or less, or 60 hours or less, for a sugar composition of50 g/l glucose and corresponding other sugars from the cellulosicmaterial (e.g. 50 g/l xylose, 35 g/l L-arabinose and 10 g/l galactose).For more dilute sugar compositions, the fermentation time maycorrespondingly be reduced. In an embodiment the fermentation time ofthe ethanol production step is between 10 and 50 hours for ethanol madeout of C6 sugars and between 20 and 100 hours for ethanol made out of C5sugars. In an embodiment the fermentation time of the succinic acidproduction step is between 20 and 70 hours.

The fermentation method may be an aerobic or an anaerobic fermentationmethod. An anaerobic fermentation method is herein defined as afermentation method run in the absence of oxygen or in whichsubstantially no oxygen is consumed, preferably less than 5, 2.5 or 1mmol/L/h, more preferably 0 mmol/L/h is consumed (i.e. oxygenconsumption is not detectable), and wherein organic molecules serve asboth electron donor and electron acceptors. In the absence of oxygen,NADH produced in glycolysis and biomass formation, cannot be oxidised byoxidative phosphorylation. To solve this problem many microorganisms usepyruvate or one of its derivatives as an electron and hydrogen acceptorthereby regenerating NAD⁺. Thus, in a preferred anaerobic fermentationmethod pyruvate is used as an electron (and hydrogen acceptor) and isreduced to fermentation products such as ethanol, lactic acid,3-hydroxy-propionic acid, acrylic acid, acetic acid, succinic acid,citric acid, malic acid, fumaric acid, an amino acid, 1,3-propane-diol,ethylene, glycerol, butanol, a β-lactam antibiotics and a cephalosporin.In a preferred embodiment, the fermentation method is anaerobic. Ananaerobic method is advantageous, since it is cheaper than aerobicmethods: less special equipment is needed. Furthermore, anaerobicmethods are expected to give a higher product yield than aerobicmethods. Under aerobic conditions, usually the biomass yield is higherthan under anaerobic conditions. As a consequence, usually under aerobicconditions, the expected product yield is lower than under anaerobicconditions.

In another embodiment, the fermentation method is under oxygen-limitedconditions. More preferably, the fermentation method is aerobic andunder oxygen-limited conditions. An oxygen-limited fermentation methodis a method in which the oxygen consumption is limited by the oxygentransfer from the gas to the liquid. The degree of oxygen limitation isdetermined by the amount and composition of the ingoing gas flow as wellas the actual mixing/mass transfer properties of the fermentationequipment used. Preferably, in a method under oxygen-limited conditions,the rate of oxygen consumption is at least 5.5, more preferably at least6 and even more preferably at least 7 mmol/L/h.

In an embodiment the alcohol fermentation method is anaerobic, while theorganic acid fermentation method is aerobic, but done underoxygen-limited conditions.

The fermentation method is preferably run at a temperature that isoptimal for the microorganism used. Thus, for most yeasts or fungalcells, the fermentation method is performed at a temperature which isless than 42° C., preferably 38° C. or lower. For yeast or filamentousfungal host cells, the fermentation method is preferably performed at atemperature which is lower than 35, 33, 30 or 28° C. and at atemperature which is higher than 20, 22, or 25° C. In an embodiment thealcohol fermentation step and the organic acid fermentation step areperformed between 25° C. and 35° C.

In an embodiment of the application, the fermentations are conductedwith a fermenting microorganism. In an embodiment of the application,the alcohol (e.g. ethanol) fermentations of C5 sugars are conducted witha C5 fermenting microorganism. In an embodiment of the application, thealcohol (e.g. ethanol) fermentations of C6 sugars are conducted with aC5 fermenting microorganism or a commercial C6 fermenting microorganism.Commercially available yeast suitable for ethanol production include,but are not limited to, BIOFERM™ AFT and XR (NABC—North AmericanBioproducts Corporation, GA, USA), ETHANOL RED™ yeast(Fermentis/Lesaffre, USA), FALI™ (Fleischmann's Yeast, USA), FERMIOL™(DSM Specialties), GERT STRAND™ (Gert Strand AB, Sweden), andSUPERSTART™ and THERMOSACC™ fresh yeast (Ethanol Technology, WI, USA).

In an embodiment propagation of the alcohol producing microorganismand/or the organic acid producing microorganism is performed in one ormore propagation containers. After propagation, the alcohol producingmicroorganism and/or the organic acid producing microorganism may beadded to one or more fermentation containers. Alternatively, thepropagation of the alcohol producing microorganism and/or the organicacid producing microorganism is combined with the fermentation by thealcohol producing microorganism and/or the organic acid producingmicroorganism to produce alcohol and/or organic acid, respectively.

In an embodiment the alcohol producing microorganism is a microorganismthat is able to ferment at least one C5 sugar. Preferably, it also isable to ferment at least one C6 sugar. In an embodiment the applicationrelates to a method for the preparation of ethanol from cellulosicmaterial, comprising the steps of (a) performing a method for thepreparation of a sugar product from cellulosic material as describedabove, (b) fermentation of the enzymatically hydrolysed cellulosicmaterial to produce ethanol; and (c) optionally, recovery of theethanol. The fermentation can be done with a microorganism that is ableto ferment at least one C5 sugar.

In an embodiment the organic acid producing microorganism is amicroorganism that is able to ferment at least one C6 sugar. In anembodiment the application relates to a method for the preparation ofsuccinic acid from cellulosic material, comprising the steps of (a)performing a method for the preparation of a sugar product fromcellulosic material as described above, (b) fermentation of theenzymatically hydrolysed cellulosic material to produce succinic acid;and (c) optionally, recovery of the succinic acid. The fermentation canbe done with a microorganism that is able to ferment at least one C6sugar.

The alcohol producing microorganisms may be a prokaryotic or eukaryoticorganism. The microorganism used in the method may be a geneticallyengineered microorganism. Examples of suitable alcohol producingorganisms are yeasts, for instance Saccharomyces, e.g. Saccharomycescerevisiae, Saccharomyces pastorianus or Saccharomyces uvarum,Hansenula, Issatchenkia, e.g. Issatchenkia orientalis, Pichia, e.g.Pichia stipites or Pichia pastoris, Kluyveromyces, e.g. Kluyveromycesfagilis, Candida, e.g. Candida pseudotropicalis or Candidaacidothermophilum, Pachysolen, e.g. Pachysolen tannophilus or bacteria,for instance Lactobacillus, e.g. Lactobacillus lactis, Geobacillus,Zymomonas, e.g. Zymomonas mobilis, Clostridium, e.g. Clostridiumphytofermentans, Escherichia, e.g. E. coli, Klebsiella, e.g. Klebsiellaoxytoca. In an embodiment the microorganism that is able to ferment atleast one C5 sugar is a yeast. In an embodiment, the yeast is belongs tothe genus Saccharomyces, preferably of the species Saccharomycescerevisiae. The yeast, e.g. Saccharomyces cerevisiae, used in themethods as described herein is capable of converting hexose (C6) sugarsand pentose (C5) sugars. The yeast, e.g. Saccharomyces cerevisiae, usedin the methods as described herein can anaerobically ferment at leastone C6 sugar and at least one C5 sugar. For example, the yeast iscapable of using L-arabinose and xylose in addition to glucoseanaerobically. In an embodiment, the yeast is capable of convertingL-arabinose into L-ribulose and/or xylulose 5-phosphate and/or into adesired fermentation product, for example into ethanol. Organisms, forexample Saccharomyces cerevisiae strains, able to produce ethanol fromL-arabinose may be produced by modifying a host yeast introducing thearaA (L-arabinose isomerase), araB (L-ribuloglyoxalate) and araD(L-ribulose-5-P4-epimerase) genes from a suitable source. Such genes maybe introduced into a host cell in order that it is capable of usingarabinose. Such an approach is given is described in WO 2003/095627.araA, araB and araD genes from Lactobacillus plantarum may be used andare disclosed in WO2008/041840. The araA gene from Bacillus subtilis andthe araB and araD genes from Escherichia coli may be used and aredisclosed in EP1499708. In another embodiment, araA, araB and araD genesmay derived from of at least one of the genus Clavibacter, Arthrobacterand/or Gramella, in particular one of Clavibacter michiganensis,Arthrobacter aurescens, and/or Gramella forsetii, as disclosed in WO2009011591. In an embodiment, the yeast may also comprise one or morecopies of xylose isomerase gene and/or one or more copies of xylosereductase and/or xylitol dehydrogenase.

The yeast may comprise one or more genetic modifications to allow theyeast to ferment xylose. Examples of genetic modifications areintroduction of one or more xylA-gene, XYL1 gene and XYL2 gene and/orXKS1-gene; deletion of the aldose reductase (GRE3) gene; overexpressionof PPP-genes TAD, TKL1, RPE1 and RKI1 to allow the increase of the fluxthrough the pentose phosphate pathway in the cell. Examples ofgenetically engineered yeast are described in EP1468093 and/orWO2006/009434.

An example of a suitable commercial yeast is RN1016 that is a xylose andglucose fermenting Saccharomyces cerevisiae strain from DSM, theNetherlands.

In an embodiment, the fermentation method for the production of ethanolis anaerobic. Anaerobic has already been defined earlier herein. Inanother preferred embodiment, the fermentation method for the productionof ethanol is aerobic. In another preferred embodiment, the fermentationmethod for the production of ethanol is under oxygen-limited conditions,more preferably aerobic and under oxygen-limited conditions.Oxygen-limited conditions have already been defined earlier herein.

The volumetric ethanol productivity is preferably at least 0.5, 1.0,1.5, 2.0, 2.5, 3.0, 5.0 or 10.0 g ethanol per litre per hour. Theethanol yield on L-arabinose and optionally xylose and/or glucose in themethod preferably is at least 20, 25, 30, 35, 40, 45, 50, 60, 70, 80,90, 95 or 98%. The ethanol yield is herein defined as a percentage ofthe theoretical maximum yield, which, for glucose and L-arabinose andoptionally xylose is 0.51 g ethanol per g glucose or xylose.

In one aspect, the fermentation method leading to the production ofethanol, has several advantages by comparison to known ethanolfermentations methods: anaerobic methods are possible; oxygen limitedconditions are possible; higher ethanol yields and ethanol productionrates can be obtained; the strain used may be able to use L-arabinoseand optionally xylose.

Alternatively to the fermentation methods described above, at least twodistinct cells may be used, this means this method is a co-fermentationmethod. All preferred embodiments of the fermentation methods asdescribed above are also preferred embodiments of this co-fermentationmethod: identity of the fermentation product, identity of source ofL-arabinose and source of xylose, conditions of fermentation (aerobic oranaerobic conditions, oxygen-limited conditions, temperature at whichthe method is being carried out, productivity of ethanol, yield ofethanol).

The organic acid producing microorganisms may be a prokaryotic oreukaryotic organism. The microorganism used in the method may be agenetically engineered microorganism. Examples of suitable organic acidproducing organisms are yeasts, for instance Saccharomyces, e.g.Saccharomyces cerevisiae; fungi for instance Aspergillus strains, suchas Aspergillus niger and Aspergillus fumigatus, Byssochlamys nivea,Lentinus degener, Paecilomyces varioti and Penicillium viniferum; andbacteria, for instance Anaerobiospirifium succiniciproducens,Actinobacillus succinogenes, Mannhei succiniciproducers MBEL 55E,Escherichia coli, Propionibacterium species, Pectinatus sp., Bacteroidessp., such as Bacteroides amylophilus, Ruminococcus flavefaciens,Prevotella ruminicola, Succcinimonas amylolytica, Succinivibriodextrinisolvens, Wolinella succinogenes, and Cytophaga succinicans. Inan embodiment the organic acid producing microorganism that is able toferment at least one C6 sugar is a yeast. In an embodiment, the yeast isbelongs to the genus Saccharomyces, preferably of the speciesSaccharomyces cerevisiae. The yeast, e.g. Saccharomyces cerevisiae, usedin the production methods of organic acid as described herein is capableof converting hexose (C6) sugars. The yeast, e.g. Saccharomycescerevisiae, used in the methods as described herein can anaerobicallyferment at least one C6 sugar.

The overall reaction time (or the reaction time of hydrolysis step andfermentation step together) may be reduced. In one embodiment, theoverall reaction time is 300 hours or less, 200 hours or less, 150 hoursor less, 140 hours or less, 130 or less, 120 hours or less, 110 hours orless, 100 hours of less, 90 hours or less, 80 hours or less, 75 hours orless, or about 72 hours at 90% glucose yield. Correspondingly, loweroverall reaction times may be reached at lower glucose yield.

Fermentation products that may be produced by the methods as describedherein can be any substance derived from fermentation. They include, butare not limited to, alcohol (such as arabinitol, butanol, ethanol,glycerol, methanol, 1,3-propanediol, sorbitol, and xylitol); organicacid (such as acetic acid, acetonic acid, adipic acid, ascorbic acid,acrylic acid, citric acid, 2,5-diketo-D-gluconic acid, formic acid,fumaric acid, glucaric acid, gluconic acid, glucuronic acid, glutaricacid, 3-hydroxypropionic acid, itaconic acid, lactic acid, maleic acid,malic acid, malonic acid, oxalic acid, oxaloacetic acid, propionic acid,succinic acid, and xylonic acid); ketones (such as acetone); amino acids(such as aspartic acid, glutamic acid, glycine, lysine, serine,tryptophan, and threonine); alkanes (such as pentane, hexane, heptane,octane, nonane, decane, undecane, and dodecane), cycloalkanes (such ascyclopentane, cyclohexane, cycloheptane, and cyclooctane), alkenes (suchas pentene, hexene, heptene, and octene); and gases (such as methane,hydrogen (H₂), carbon dioxide (C0₂), and carbon monoxide (CO)). Thefermentation product can also be a protein, a vitamin, a pharmaceutical,an animal feed supplement, a specialty chemical, a chemical feedstock, aplastic, a solvent, ethylene, an enzyme, such as a protease, acellulase, an amylase, a glucanase, a lactase, a lipase, a lyase, anoxidoreductase, a transferase or a xylanase. In a preferred embodimentan organic acid and/or an alcohol is prepared in the fermentationmethods as described herein. In a preferred embodiment succinic acidand/or ethanol is prepared in the fermentation methods as describedherein.

Use of the Polypeptide and Composition as Described Herein

The variant polypeptides and compositions as described herein may beused in many different applications. For instance, they may be used toproduce fermentable sugars. The fermentable sugars can then, as part ofa biofuel method, be converted into biogas or ethanol, butanol,isobutanol, 2-butanol or other fermentation products. A non-extendiblelist is given above.

By “fermentable sugars” is meant sugars which can be consumed by amicroorganism or converted by a microorganism into a fermentationproduct.

Alternatively, a variant polypeptide as described herein and/or acomposition as described herein may be used in the production of a foodproduct, a detergent composition, in the paper and pulp industry, inantibacterial formulations, in pharmaceutical products to name just afew. Some of the uses will be illustrated in more detail below.

In the uses and methods described below, the components of thecompositions described above may be provided concomitantly (i.e. as asingle composition per se) or separately or sequentially.

In principle, a variant polypeptide as described herein and/orcomposition as described herein may be used in any method which requiresthe treatment of a material which comprises polysaccharide. Thus, avariant polypeptide and/or composition as described herein may be usedin the treatment of polysaccharide material. Herein, polysaccharidematerial is a material which comprises or consists essential of one or,more typically, more than one polysaccharide.

The application also provides use of a polypeptide and/or composition adescribed herein in a method for the preparation of biogas. Biogastypically refers to a gas produced by the biological breakdown oforganic matter, for example cellulosic material, in the absence ofoxygen. Biogas originates from biogenic material and is a type ofbiofuel. One type of biogas is produced by anaerobic digestion orfermentation of biodegradable materials such as biomass, manure orsewage, municipal waste, and energy crops. This type of biogas iscomprised primarily of methane and carbon dioxide. The gas methane canbe combusted or oxidized with oxygen. Air contains 21% oxygen. Thisenergy release allows biogas to be used as a fuel. Biogas can be used asa low-cost fuel in any country for any heating purpose, such as cooking.It can also be utilized in modern waste management facilities where itcan be used to run any type of heat engine, to generate eithermechanical or electrical power. The first step in microbial biogasproduction consists in the enzymatic degradation of polymers and complexsubstrates. Accordingly, the application provides a method forpreparation of a biogas in which a cellulosic substrate is contactedwith a polypeptide and/or composition as described herein, thereby toyield fermentable material which may be converted into a biogas by anorganism, such as a microorganism. In such a method, a polypeptideand/or composition as described herein may be provided by way of anorganism, for example a microorganism which expresses a polypeptideand/or composition as described herein.

The polypeptides and/or compositions as described herein may be used ina method of treating material to degrade or modify the cellulose and/orhemicellulose and/or pectic substance constituents of the material. Suchmethods may be useful in the preparation of a food product. Accordingly,the application provides a method for preparing a food product whichmethod comprises incorporating a variant polypeptide and/or compositionas described herein during preparation of the food product. Theapplication also provides a method of methoding a cellulosic material,which method comprises contacting the cellulosic material with a variantpolypeptide and/or composition as described herein to degrade or modifythe cellulose in the material. The present application also provides amethod for reducing the viscosity, clarity and/or filterability of acellulosic material, which method comprises contacting the material witha variant polypeptide and/or composition as described herein in anamount effective in degrading cellulose and/or hemicellulose and/orpectic substances in the material. Cellulosic materials in this respectinclude, but are not limited to, plant pulp, parts of plants and plantextracts. In the context of this application an extract from a plantmaterial is any substance which can be derived from plant material byextraction (mechanical and/or chemical), methoding or by otherseparation techniques. The extract may be juice, nectar, base orconcentrate made thereof. The plant material may comprise or be derivedfrom vegetables (e.g. carrots, celery, onions, legumes or leguminousplants (soy, soybean, peas)) or fruit (e.g., pome or seed fruit (apples,pears, quince etc.), grapes, tomatoes, citrus (orange, lemon, lime,mandarin), melons, prunes, cherries, black currants, redcurrants,raspberries, strawberries, cranberries, pineapple and other tropicalfruits), trees and parts thereof (e.g. pollen, from pine trees), orcereal (oats, barley, wheat, maize, rice). The material (to behydrolysed) may also be agricultural residues, such as sugar beet pulp,corn cobs, wheat straw, (ground) nutshells, or recyclable materials,e.g. (waste) paper. The variant polypeptides as described herein canthus be used to treat plant material including plant pulp and plantextracts. They may also be used to treat liquid or solid foodstuffs oredible foodstuff ingredients, or be used in the extraction of coffee,plant oils, starch or as a thickener in foods. Typically, the variantpolypeptides as described herein are used as a composition as describedabove. The composition will generally be added to plant pulp obtainableby, for example mechanical methoding such as crushing or milling plantmaterial. Incubation of the composition with the plant will typically becarried out for at time of from 10 minutes to 5 hours. The methodingtemperature is preferably from about 10° C. to about 55° C. and one canuse from about 10 g to about 300 g of enzyme per ton of material to betreated. The variant polypeptides or compositions as described hereinmay be added sequentially or at the same time to the plant pulp.Depending on the composition of the enzyme preparation the plantmaterial may first be macerated (e.g. to a pure) or liquefied. Using thevariant polypeptides as described herein, methoding parameters such asthe yield of the extraction, viscosity of the extract and/or quality ofthe extract can be improved. Alternatively, or in addition to the above,a variant polypeptide and/or composition as described herein may beadded to the raw juice obtained from pressing or liquefying the plantpulp. Treatment of the raw juice will be carried out in a similar mannerto the plant pulp in respect of dosage, temperature and holding time.Again, other enzymes such as those discussed previously may be included.Typical incubation conditions are as described above. Once the raw juicehas been incubated with the variant polypeptides or compositions asdescribed herein, the juice is then centrifuged or (ultra) filtered toproduce the final product. After treatment with the variant polypeptideand/or composition as described herein, the (end) product can be heattreated, e.g. at about 100° C. for a time of from about 1 minute toabout 1 hour, under conditions to partially or fully inactivate thevariant polypeptide and/or composition as described herein. A variantpolypeptide and/or composition as described herein may also be usedduring the preparation of fruit or vegetable purees. The variantpolypeptide and/or composition as described herein may also be used inbrewing, wine making, distilling or baking. It may therefore be used inthe preparation of alcoholic beverages such as wine and beer. Forexample, it may improve the filterability or clarity, for example ofbeers, wort (e.g. containing barley and/or sorghum malt) or wine.Furthermore, a variant polypeptide and/or composition as describedherein may be used for treatment of brewers spent grain, i.e. residualsfrom beer wort production containing barley or malted barley or othercereals, so as to improve the utilization of the residuals for e.g.animal feed. A variant polypeptide and/or composition as describedherein may assist in the removal of dissolved organic substances frombroth or culture media, for example where distillery waste from organicorigin is bioconverted into microbial biomass. The variant polypeptideand/or composition as described herein may improve filterability and/orreduce viscosity in glucose syrups, such as from cereals produced byliquefaction (e.g. with α-amylase). In baking, the variant polypeptideand/or composition as described herein may improve the dough structure,modify its stickiness or suppleness, improve the loaf volume and/orcrumb structure or impart better textural characteristics such as break,shred or crumb quality. The present application thus relates to methodsfor preparing a dough or a cereal-based food product comprisingincorporating into the dough a variant polypeptide and/or composition asdescribed herein. This may improve one or more properties of the doughor the cereal-based food product obtained from the dough relative to adough or a cereal-based food product in which the variant polypeptideand/or composition is not incorporated. The preparation of thecereal-based food product further can comprise steps known in the artsuch as boiling, drying, frying, steaming or baking of the obtaineddough. Products that are made from a dough that is boiled are forexample boiled noodles, dumplings, products that are made from frieddough are for example doughnuts, beignets, fried noodles, products thatare made for steamed dough are for example steamed buns and steamednoodles, examples of products made from dried dough are pasta and driednoodles and examples of products made from baked dough are bread,cookies and cake. The term “improved property” is defined herein as anyproperty of a dough and/or a product obtained from the dough,particularly a cereal-based food product, which is improved by theaction of the variant polypeptide and/or composition as described hereinrelative to a dough or product in which the variant polypeptide and/orcomposition as described herein is not incorporated. The improvedproperty may include, but is not limited to, increased strength of thedough, increased elasticity of the dough, increased stability of thedough, improved machinability of the dough, improved proofing resistanceof the dough, reduced stickiness of the dough, improved extensibility ofthe dough, increased volume of the cereal-based food product, reducedblistering of the cereal-based food product, improved crumb structure ofthe baked product, improved softness of the cereal-based food product,improved flavour of the cereal-based food product, improved anti-stalingof the cereal-based food product. Improved properties related to pastaand noodle type of cereal-based products are for example improvedfirmness, reduced stickiness, improved cohesiveness and reduced cookingloss. Non-starch polysaccharides (NSP) can increase the viscosity of thedigesta which can, in turn, decrease nutrient availability and animalperformance. Adding specific nutrients to feed improves animal digestionand thereby reduces feed costs. Non-starch polysaccharides (NSPs) arealso present in virtually all feed ingredients of plant origin. NSPs arepoorly utilized and can, when solubilized, exert adverse effects ondigestion. Exogenous enzymes can contribute to a better utilization ofthese NSPs and as a consequence reduce any anti-nutritional effects. Avariant polypeptide and/or composition as described herein can be usedfor this purpose in cereal-based diets for poultry and, to a lesserextent, for pigs and other species.

A variant polypeptide and/or composition as described herein may be usedin the detergent industry, for example for removal from laundry ofcarbohydrate-based stains. A detergent composition may comprise avariant polypeptide and/or composition as described herein and, inaddition, one or more of a cellulase, a hemicellulase, a pectinase, aprotease, a lipase, a cutinase, an amylase or a carbohydrase. Adetergent composition comprising a variant polypeptide and/orcomposition as described herein may be in any convenient form, forexample a paste, a gel, a powder or a liquid. A liquid detergent may beaqueous, typically containing up to about 70% water and from about 0 toabout 30% organic solvent or non-aqueous material. Such a detergentcomposition may, for example, be formulated as a hand or machine laundrydetergent composition including a laundry additive composition suitablefor pre-treatment of stained fabrics and a rinse added fabric softenercomposition, or be formulated as a detergent composition for use ingeneral household hard surface cleaning operations, or be formulated forhand or machine dish washing operations. In general, the properties ofthe variant polypeptide and/or composition as described herein should becompatible with the selected detergent (for example, pH-optimum,compatibility with other enzymatic and/or non-enzymatic ingredients,etc.) and the variant polypeptide and/or composition as described hereinshould be present in an effective amount. A detergent composition maycomprise a surfactant, for example an anionic or non-ionic surfactant, adetergent builder or complexing agent, one or more polymers, a bleachingsystem (for example an H₂O₂ source) or an enzyme stabilizer. A detergentcomposition may also comprise any other conventional detergentingredient such as, for example, a conditioner including a clay, a foambooster, a sud suppressor, an anti-corrosion agent, a soil-suspendingagent, an an-soil redeposition agent, a dye, a bactericide, an opticalbrightener, a hydrotropes, a tarnish inhibitor or a perfume.

A variant polypeptide and/or composition as described herein may be usedin the paper and pulp industry, inter alia in the bleaching method toenhance the brightness of bleached pulps whereby the amount of chlorineused in the bleaching stages may be reduced, and to increase thefreeness of pulps in the recycled paper method. Furthermore, a variantpolypeptide and/or composition as described herein may be used fortreatment of lignocellulosic pulp so as to improve the bleachabilitythereof. Thereby the amount of chlorine need to obtain a satisfactorybleaching of the pulp may be reduced.

A variant polypeptide and/or composition as described herein may be usedin a method of reducing the rate at which cellulose-containing fabricsbecome harsh or of reducing the harshness of cellulose-containingfabrics, the method comprising treating cellulose-containing fabricswith a variant polypeptide and/or composition as described above. Thepresent application further relates to a method providing colourclarification of coloured cellulose-containing fabrics, the methodcomprising treating coloured cellulose-containing fabrics with a variantpolypeptide and/or composition as described above, and a method ofproviding a localized variation in colour of colouredcellulose-containing fabrics, the method comprising treating colouredcellulose-containing fabrics with a variant polypeptide and/orcomposition as described above. The methods as described herein may becarried out by treating cellulose-containing fabrics during washing.However, if desired, treatment of the fabrics may also be carried outduring soaking or rinsing or simply by adding the polypeptide and/orcomposition as described above to water in which the fabrics are or willbe immersed.

In addition, a variant polypeptide and/or composition as describedherein can also be used in antibacterial formulation as well as inpharmaceutical products such as throat lozenges, toothpastes, andmouthwash.

EXAMPLES Experimental Information Strains

WT 1: Aspergillus niger strain was deposited at the CENTRAAL BUREAU VOORSCHIMMELCULTURES, Uppsalalaan 8, P.O. Box 85167, NL-3508 AD Utrecht, TheNetherlands on 10 Aug. 1988 under the deposit number CBS 513.88.

WT 2: This A. niger strain is a WT 1 strain comprising a deletion of thegene encoding glucoamylase (glaA). WT 2 was constructed by using the“MARKER-GENE FREE” approach as described in EP0635574B1. In this patentit is extensively described how to delete glaA specific DNA sequences inthe genome of CBS 513.88. The procedure resulted in a MARKER-GENE FREEΔglaA recombinant A. niger CBS 513.88 strain, possessing finally noforeign DNA sequences at all.

WT 3: This A. niger strain is a WT 2 strain comprising a deletion of thepepA gene encoding the major extracellular aspartic protease PepA, asdescribed by van den Hombergh et al. (van den Hombergh J P, SollewijnGelpke M D, van de Vondervoort P J, Buxton F P, Visser J.(1997)—Disruption of three acid proteases in Aspergillus niger-effectson protease spectrum, intracellular proteolysis, and degradation oftarget proteins—Eur J Biochem. 247(2): 605-13). The procedure resultedin a MARKER-GENE FREE WT 3 strain with the pepA gene inactivated in theWT 2 strain background.

Suitable Rasamsonia (Talaromyces) emersonii strains to show the effectand advantages as described herein are for example TEC-101, TEC-147,TEC-192, TEC-201 or TEC-210. They are described in WO 2011/000949. The“4E mix” or “4E composition” containing CBHI, CBHII, EG4 andBETA-GLUCOSIDASE (30 wt %, 25 wt %, 28 wt % and 8 wt %, respectively, wt% on dry matter protein) has been described in WO 2011/098577.

Rasamsonia (Talaromyces) emersonii strain TEC-101 (also designated asFBG 101) was deposited at CENTRAAL BUREAU VOOR SCHIMMELCULTURES,Uppsalalaan 8, P.O. Box 85167, NL-3508 AD Utrecht, The Netherlands on 30Jun. 2010 having the Accession Number CBS 127450.

Molecular Biology Techniques

In the above strains, using molecular biology techniques known to theskilled person (see: Sambrook & Russell, Molecular Cloning: A LaboratoryManual, 3rd Ed., CSHL Press, Cold Spring Harbor, N.Y., 2001), severalgenes are overexpressed and others are down regulated as describedbelow. Examples of the general design of expression vectors for geneoverexpression and disruption vectors for down-regulation,transformation, use of markers and selective media can be found in WO1998/46772, WO 1999/32617, WO 2001/121779, WO 2005/095624, WO2006/040312, EP0635574B, WO 2005/100573, WO 2011/009700, WO 2012/001169and WO 2011/054899. All gene replacement vectors comprise approximately1-2 kb flanking regions of the respective Open Reading Frame (ORF)sequences, to target for homologous recombination at the predestinedgenomic loci. In addition, A. nigervectors contain the A. nidulansbi-directional amdS selection marker for transformation, in betweendirect repeats. The method applied for gene deletion uses linear DNA,which integrates into the genome at the homologous locus of the flankingsequences by a double cross-over, thus substituting the gene to bedeleted by the amdS gene. After transformation, the direct repeats allowfor the removal of the selection marker by a (second) homologousrecombination event. The removal of the amdS marker has been done byplating on fluoro-acetamide media, resulting in the selection ofmarker-gene-free strains. Using this strategy of transformation andsubsequent counter-selection, which is also described as the“MARKER-GENE FREE” approach in EP 0 635 574. The amdS marker can be usedindefinitely in strain modification programs.

Media and Solutions

Potato dextrose agar, PDA, (Fluke, Cat. No. 70139): per litre: Potatoextract 4 g; Dextrose 20 g; Bacto agar 15 g; pH 5.4; Sterilize 20 min at120° C.

Rasamsonia agar medium: per litre: Salt fraction no. 3 15 g; Cellulose30 g; Bacto peptone 7.5 g; Grain flour 15 g; KH₂PO₄ 5 g; CaCl₂.2aq 1 g;Bacto agar 20 g; pH 6.0; Sterilize 20 min at 120° C.

Salt fraction composition: The “salt fraction no. 3” is fitting thedisclosure of WO 98/37179, Table 1. Deviations from the composition ofthis table are CaCl₂.2aq 1.0 g/l, KCl 1.8 g/L, citric acid 1 aq 0.45 g/L(chelating agent).

Rasamsonia shake flask medium 1: per litre: Glucose 20 g; Yeast extract(Difco) 20 g; Clerol FBA3107 (AF) 4 drops; MES 30 g; pH 6.0; Sterilize20 min at 120° C.

Rasamsonia shake flask medium 2: per litre: Salt fraction no. 3 10 g;glucose

10 g; KH₂PO₄. 5 g; NaH₂PO₄ 2 g; (NH₄)₂SO₄. 5 g; MES 30 g; pH 5.4;Sterilize 20 min at 120° C.

Rasamsonia shake flask medium 3: per litre: Salt fraction no. 3 10 g;cellulose 20 g; KH₂PO₄ 5 g; NaH₂PO₄ 2 g; (NH₄)₂SO₄ 5 g; MES 30 g; pH5.4; Sterilize 20 min at 120° C.

Rasamsonia shake flask medium 4: per litre: Salt fraction no. 3 10 g;cellulose

15 g; glucose 5 g; KH₂PO₄ 5 g; NaH₂PO₄ 2 g; (NH₄)₂SO₄ 5 g; MES 30 g; pH5.4; Sterilize 20 min at 120° C.

Spore Batch Preparation for Rasamsonia

Strains were grown from stocks on Rasamsonia agar medium in 10 cmdiameter Petri dishes for 5-7 days at 40° C. For MTP fermentations,strains were grown in 96-well plates containing Rasamsonia agar medium.Strain stocks are stored at −80° C. in 10% glycerol.

Chromosomal DNA Isolation

Strains were grown in YGG medium (per liter: 8 g KCl, 16 g glucose.H₂O,20 ml of 10% yeast extract, 10 ml of 100× pen/strep, 6.66 g YNB+aminoacids, 1.5 g citric acid, and 6 g K₂HPO₄) for 16 hours at 42° C., 250rpm, and chromosomal DNA was isolated using the DNeasy plant mini kit(Qiagen, Hilden, Germany).

Shake Flask Growth Protocol of Rasamsonia

Spores were inoculated into 100 ml shake flasks containing 20 ml ofRasamsonia shake flask medium 1 and incubated at 45° C. at 250 rpm in anincubator shaker for 1 day (preculture 1) and 1 or 2 ml of biomass frompreculture 1 was transferred to 100 ml shake flasks containing 20 ml ofRasamsonia shake flask medium 2 and grown under conditions as describedabove for 1 day (preculture 2). Subsequently, 1 or 2 ml of biomass frompreculture 2 was transferred to 100 ml shake flasks containing 20 ml ofRasamsonia shake flask medium 3 or 4 and grown under conditionsdescribed above for 3 days.

Protein Analysis

Protein samples are separated under reducing conditions on NuPAGE 4-12%Bis-Tris gel (Invitrogen, Breda, The Netherlands) and stained. Gels arestained with either InstantBlue (Expedeon, Cambridge, United Kingdom),SimplyBlue safestain (Invitrogen, Breda, The Netherlands) or Sypro Ruby(Invitrogen, Breda, The Netherlands) according to manufacturer'sinstructions.

Total Protein Concentration Determination with TCA-Biuret Method

Concentrated protein samples (supernatants) were diluted with water to aconcentration between 2 and 8 mg/ml. Bovine serum albumin (BSA)dilutions (0, 1, 2, 5, 8 and 10 mg/ml) were made and included as samplesto generate a calibration curve. Of each diluted protein sample, 270 μlwas transferred into a 10 ml tube containing 830 μl of a 12% (w/v)trichloro acetic acid solution in aceton and mixed thoroughly.Subsequently, the tubes were incubated on ice water for one hour andcentrifuged for 30 minutes, at 4° C. and 6000 rpm. The supernatant wasdiscarded and pellets were dried by inverting the tubes on a tissue andletting them stand for 30 minutes at room temperature. Next, 3 mlBioQuant Biuret reagent mix was added to the pellet in the tube and thepellet was solubilised upon mixing. Next, 1 ml water was added to thetube, the tube was mixed thoroughly and incubated at room temperaturefor 30 minutes. The absorption of the mixture was measured at 546 nmwith a water sample used as a blank measurement and the proteinconcentration was calculated via the BSA calibration line.

Cellulase Activity Assays

In order to measure cellulase activity, corn stover activity assays areperformed. Cellulase activity is measured in supernatants (the liquidpart of the broth wherein the cells were cultured) of an empty strainand the transformant:

Preparation of Pretreated, Corn Stover Substrate.

Dilute-acid pre-treated corn stover is obtained as described in Schell,D. J., Applied Biochemistry and Biotechnology (2003), vol. 105-108, pp69-85. A pilot scale pretreatment reactor is used operating at steadystate conditions of 190° C., 1 min residence time and an effective H₂SO₄acid concentration of 1.45% (w/w) in the liquid phase.

Assay 1: Microtiter Plate (MTP) 2% Unwashed Acid Pretreated Corn-StoverSugar-Release Assay in which Supernatants are Spiked on Top of TEC-210or 4E Mix

For each (hemi-)cellulase assay, the stored samples are analyzed twice;100 μL of sample (e.g. shake flask supernatant) and 100 μl of a(hemi-)cellulase base mix (3.5 mg/g DM TEC-210 or a 4 enzyme mix at atotal dosage of 3.5 mg/g DM consisting of 0.3 mg/g DM BG (9% of totalprotein 4E mix), 1 mg/g DM CBHI (30% of total protein 4E mix), 0.9 mg/gDM CBHII (25% of total protein 4E mix) and 1.3 mg/g DM GH61 (36% oftotal protein 4E mix)) is transferred to two suitable vials: one vialcontaining 800 μL 2.5% (w/w) dry matter of the acid pretreated cornstover in a 50 mM citrate buffer, buffered at pH 4.5. The other vialconsisted of a blank, where the 800 μL 2.5% (w/w) dry matter, acidpretreated corn stover is replaced by 800 μL 50 mM citrate buffer,buffered at pH 4.5. The assay samples are incubated for 72 hrs at 65° C.After incubation of the assay samples, a fixed volume of D₂O (with 0.5g/L DSS) containing an internal standard (maleic acid (20 g/L) and EDTA(40 g/L)) is added. The amount of sugar released, is based on the signalbetween 4.65-4.61 ppm, relative to DSS, and is determined by means of 1D1H NMR operating at a proton frequency of 500 MHz, using a pulseprogramwith water suppression, at a temperature of 27° C.

The (hemi)-cellulase enzyme solution may contain residual sugars.Therefore, the results of the assay are corrected for the sugar contentmeasured after incubation of the enzyme solution.

Assay 2: Dose-Response 2% Unwashed Acid Pretreated Corn Stover SugarRelease Assay in MTP

Since glucose release by cellulases is not a linear function of thequantity of enzyme in the composition, in other words, twice the amountof enzyme does not automatically result in twice the amount of glucoseat a fixed time point. Therefore, the activity of the cellulose enzymemixture is assessed in a dose response based assay, in which the dosageis based on equal amount of protein per cellulose mixture tested.

Overall cellulase activity of the mixture measured with unwashed acidpretreated corn stover as substrate. The frozen enzyme samples arethawed and a series of 6 dilutions is made ranging from undiluted insteps of two-fold up to 32-fold in 50 mM citrate buffer pH 4.5.

200 μl of sample is transferred to a vial containing 800 μL of 2.5%(w/w) dry matter of the acid pretreated corn stover in 50 mM citratebuffer, buffered at pH 4.5. Another 200 μl sample is transferred to avial, referred to as blank, containing 800 μl of 50 mM citrate buffer,buffered at pH 4.5. In addition, a sugar background of corn stover isdetermined by incubating 800 μL 2.5% (w/w) dry matter of the acidpretreated corn stover in 50 mM citrate buffer, buffered at pH 4.5 with200 μl of 50 mM citrate buffer. All vials are incubated for 72 hr at 65°C.

After incubation, 100 μl of internal standard solution (20 g/L maleicacid, 40 g/L EDTA in D₂O) is added to the vials. All vials containingpretreated corn stover are centrifuged for 30 minutes at 5300×g and,subsequently, 600 μl of the supernatant is transferred to a new vialcontaining 400 μI of H₂O/D₂O 9:1.

The 1D ¹H-NMR spectra are recorded on an Avance III Bruker operating ata proton frequency of 500 MHz, using a pulse program with watersuppression, at a temperature of 27° C. Glucose quantification(arbitrary units) is performed based on the signal at 5.20 ppm, relativeto 4,4-Dimethyl-4-silapentane sulfonic acid with relation to theinternal standard signal at 6.30 ppm. The relative glucose release(ΔGlc) is calculated by correcting the glucose measured in the samplesby the residual sugar present in the enzyme solution (measured from theblank) and the residual sugar present in the acid pretreated cornstover.

Since the protein concentration of the samples is known, the sugarrelease can be depicted as a function of protein mg/ml of the testeddiluted sample versus the relative glucose release at time point 72hours.

Beta-Glucosidase Basic Activity Assay

The beta-glucosidase activity of the variant polypeptides was analyzedin an MTP scale activity assay using p-nitrophenyl-β-D-glucopyranosideas substrate. Rasamsonia emersonii beta-glucosidase having the aminoacid sequence of SEQ ID NO: 2 (wild-type/parent beta-glucosidase) wasused as reference. In a total volume of 100 μl, the supernatants of theA. niger MTP fermentations expressing the variant polypeptides and thewild-type beta-glucosidase are incubated with 3 mMp-nitrophenyl-β-D-glucopyranoside in 50 mM acetate buffer pH 4.5 for 10minutes at 60° C. After the incubation time, 100 μl of stop solution wasadded (1 M sodium carbonate) and the hydrolyzed free p-nitrophenol wasdetermined by measuring the absorbance at 405 nm. As a blank, thesubstrate p-nitrophenyl-β-D-glucopyranoside was incubated in 100 μl 50mM acetate buffer pH 4.5 at the same conditions (without enzyme) and theabsorbance at 405 nm was determined after addition of the stop solution.

Beta-Glucosidase Glucose Inhibition Assay

A beta-glucosidase activity assay on cellobiose substrate was performedas described above on the A. niger strains expressing the variantpolypeptides and the wild-type beta-glucosidase in the absence orpresence of added glucose to test for glucose tolerance. All assays wereperformed in 500 μl reaction volume (50 mM citrate buffer, pH 4.5) ofwhich 50 μI was a prediluted normalized beta-glucosidase enzyme sample.The amount of cellobiose substrate was 10 g/l, samples with addedglucose were typically supplemented with 20 g/l glucose, but this couldrange between 10 g/l and 200 g/l. After 20 minutes of incubation at 65°C., 100 μl of stop solution was added (1 M NaOH). The amount ofcellobiose that was converted was measured by ¹H-NMR. For each thevariant polypeptides and for the wild-type beta-glucosidase, the ratioof the cellobiose conversion was determined in the absence and in thepresence of added glucose. The ratio values were corrected for the levelof substrate conversion. The corrected activity ratio values were usedas indicators for glucose inhibition and the variant polypeptides with amore favourable glucose tolerance as compared to the beta-glucosidasehaving the amino acid sequence of SEQ ID NO: 2 (wild-type/parentbeta-glucosidase) were considered to be more resistant against glucoseinhibition.

Alternatively, the beta-glucosidase activity assay is performed asdescribed in Example 2.

Example 1 Construction of A. Niger Expression Vectors and Expression ofVariant Polypeptides

This example describes the construction of an expression construct foroverexpression of the variant polypeptides in A. niger and theexpression of the variant polypeptides.

Construction of Expression Plasmids

The sequence of the wild-type polypeptide and a set of polypeptidevariants as detailed below in Example 2 were synthesized as DNAfragments, subcloned, and sequence verified by sequence analysis.Subsequently, all variants were cloned into the pGBTOP vector (see FIG.1 for general layout) using EcoRI and PacI sites, comprising theglucoamylase promoter and terminator sequence. The translationalinitiation sequence of the glucoamylase glaA promoter has been modifiedinto 5′-CACCGTCAAA ATG-3′ and an optimal translational terminationsequence 5′-TAAA-3′ was used in the generation of the expressionconstruct (as also detailed in WO 2006/077258). The construction,general layout and use of such a vector is described in detail in WO1999/32617. The E. coli part was removed by NotI digestion prior totransformation of A. niger WT 3.

Transformation of A. Niger and Shake Flask Fermentations

A. niger strain WT 3 was co-transformed with the pGBTOP expressionconstructs and pGBAAS-1 selection marker (but any appropriate selectionmarker—vide supra: amdS, hygromycin or phleomycin or altenative markerscould be used) containing plasmid according to method described asdescribed in for example WO 1999/32617 and WO 2011/009700 (andreferences therein), and selected on acetamide containing media andcolony purified according to standard procedures, essentially asdescribed in WO 98/46772 and WO 99/32617 and references therein and inthe experimental information section herein. Strains containing theexpression constructs were selected via PCR to verify presence of thepGBTOP expression cassette. Of recombinant strains containing thepolynucleotide encoding the variant polypeptides and control A. nigerstrains, a large batch of spores was generated by plating spores ormycelia onto PDA plates (Potato Dextrose Agar, Oxoid), preparedaccording to manufacturer's instructions. After growth for 3-7 days at30° C., spores were collected after adding 0.01% Triton X-100 to theplates. After washing with sterile water, about 10⁷ spores of selectedtransformants and control strains were inoculated into 100 ml shakeflasks with baffles containing 20 ml of liquid pre-culture mediumconsisting of per liter: 30 g maltose.H₂O; 5 g yeast extract; 10 ghydrolyzed casein; 1 g KH₂PO₄; 0.5 g MgSO₄.7H₂O; 0.03 g ZnCl₂; 0.02 gCaCl₂); 0.01 g MnSO₄.4H₂O; 0.3 g FeSO₄.7H₂O; 3 g Tween 80; 10 mlpenicillin (5000 IU/ml)/Streptomycin (5000 UG/ml); pH 5.5. Thesecultures were grown at 34° C. for 16-24 hours. 10 ml of this culture wasinoculated into 500 ml shake flasks with baffles containing 100 mlfermentation medium consisting of per liter: 70 g glucose.H₂O; 25 ghydrolyzed casein; 12.5 g yeast extract; 1 g KH₂PO₄; 2 g K₂SO₄; 0.5 gMgSO₄.7H₂O; 0.03 g ZnCl₂; 0.02 g CaCl₂; 0.01 g MnSO₄.4H₂O; 0.3 gFeSO4.7H₂O; 10 ml penicillin (5000 IU/ml)/Streptomycin (5000 UG/ml);adjusted to pH 5.6. These cultures were grown at 34° C. until allglucose was depleted (usually after 4-7 days). Samples taken from thefermentation broth were centrifuged (10 minutes at 5000×g) in a swingingbucket centrifuge and supernatants collected and filtered over a 0.2 μmfilter (Nalgene).

Supernatants were analysed for expression of the variant polypeptides bySDS-PAGE and total protein measurements. A. niger supernatantscontaining the variant polypeptides are spiked on top of TEC-210 or4E-mix and analysed in a corn stover activity assay. Spiking ofsupernatant of the variant polypeptides shows increased hydrolysis ofcorn stover compared to controls in which the supernatant of the variantpolypeptides as described herein is not spiked in. The supernatants weresubjected to a MTP scale activity assay as described in Example 2 and alarge scale activity assay as described in Example 3 to identify variantpolypeptides that are less sensitive to glucose inhibition than theparent polypeptide.

Example 2

Identification of Variant Polypeptides with a Higher Glucose Tolerancein MTP Scale

All variant polypeptides were expressed in A. niger and grown in ashakeflask culture. The beta-glucosidase activity of the variantpolypeptides was analyzed in an MTP scale activity assay using 3 mMp-nitrophenyl-ß-D-glucopyranoside as substrate. Rasamsonia emersoniibeta-glucosidase having the amino acid sequence of SEQ ID NO: 2(wild-type/parent beta-glucosidase) was used as reference. Typically, 50μl of pre-diluted A. niger shakeflask fermentation material wasincubated with 50 μl 6 mM p-nitrophenyl-ß-D-glucopyranoside substratesolution in 100 mM sodium acetate buffer at pH 4.5 and at a temperatureof 60°. The dilution of the fermentation material was chosen to allow anabsorbance measurement at 405 nm in the linear range of the4-nitrophenol standard calibration curve. The incubation time of thesupernatant and the substrate was 15 minutes. After 15 minutes, thehydrolysis conversion was stopped by addition of 100 μl 1 M sodiumcarbonate stop solution and the hydrolyzed free p-nitrophenol wasdetermined by measuring the absorbance at 405 nm. As a blank, thesubstrate p-nitrophenyl-ß-D-glucopyranoside was incubated in 100 μl 50mM acetate buffer pH 4.5 at the same conditions (without enzyme) and theabsorbance at 405 nm was determined after addition of the stop solution.For each fermentation sample, the catalytic activity was determinedbased on the amount of 4-nitrophenol that was formed during theincubation per mg of protein present in the incubation. Theconcentration of 4-nitrophenol formed, was determined by measuring theabsorbance at 405 nm at basic pH and calculating the concentration viathe 4-nitrophenol standard calibration curve. Activity values per mg ofprotein were used for normalizing the variant polypeptides to the sameactivity value, which allowed activity measurements within the detectionlimit for the follow-up experiment.

For the follow-up experiment, five dilutions were made: 2-fold, 4-fold,8-fold, 16-fold and 32-fold. The beta-glucosidase activity of thevariant polypeptides and the wild-type beta-glucosidase was furtheranalyzed in a MTP scale activity assay using cellobiose as substrate andmonitoring cellobiose hydrolysis via glucose formation with NMR of theundiluted sample and the five dilutions. The same assay was performedfor all dilutions in presence of 20 g/l glucose to determine the glucosetolerance. In a total volume of 500 μl, 50 μl of the dilutedsupernatants of the A. niger MTP fermentations expressing the variantpolypeptides or the wild-type beta-glucosidase were incubated with 10g/l cellobiose with and without the addition of 20 g/l glucose in 50 mMcitrate buffer pH 4.5 for 20 minutes at 65° C. Measurements (with andwithout 20 g/l glucose) were performed for the variant polypeptides andthe wild-type beta-glucosidase. After the incubation time of 20 minutes,50 μl of a 14 M sodium hydroxide solution was added to stop the reactionvia a sudden increase of the pH. Subsequently, cellobiose and glucoselevels in the samples were analyzed with NMR.

After enzymatic incubation, 100 μl of internal standard solutioncontaining 20 g/l maleic acid and EDTA (40 g/L) in demineralized water,was added to each sample. Subsequently, the samples were lyophilizedovernight. The dried residue was dissolved in 600 μL of D₂O (with 0.5g/l DSS (4,4-dimethyl-4-silapentane-1-sulfonic acid)). 1D ¹H NMR spectrawere recorded on a Bruker Avance III HD 500 MHz, equipped with a Hecooled cryo-probe, using a pulse program without water suppression at atemperature of 29° C. with a 90 degrees excitation pulse, acquisitiontime of 2.7 s and relaxation delay of 30 s. The analyte concentrations(in g/L) were calculated based on the following signals (δ relative toDSS): Cellobiose: H1′ cellobiose peak at 4.50 ppm (d, 1H, J=8 Hz);Glucose: α-H1 glucose/α-H1 cellobiose peak at 5.22 ppm (d, 0.38H, J=4Hz). The glucose concentration was determined after correction forcellobiose. The signal user for the standard: Maleic acid peak at 6.0ppm (s, 2H). As the enzyme solutions may contain residual sugars,glucose results were corrected for the sugar content measured afterincubation of the enzyme solution. In addition, a negative control wasincluded; the cellobiose solution without added enzyme was incubated atthe same conditions and the cellobiose level was determined with NMR (nocellobiose hydrolysis should occur).

Next, the variant polypeptides with reduced glucose inhibition wereselected. The kinetics of the variants and the wild-typebeta-glucosidase were studied by measuring the cellobiose to glucoseconversion via NMR after a 20 minute incubation at 6 variantconcentrations in presence and absence of intitial glucose (20 g/L) asdescribed above. In total 12 experiments were performed per enzymevariant (six dilutions, with and without glucose). A computational modelwas applied to extract information about the kinetics of the vatriantsfrom the obtained glucose and cellobiose concentrations. The model wasdefined as follows:

BG activity was modeled as irreversible Michaelis-Menten kinetics withcompetitive glucose inhibition:

${rateBG} = \frac{{{kcat}\lbrack E\rbrack}\lbrack{cellobiose}\rbrack}{{cellobiose} + {K_{M}\left( {1 + \frac{glucose}{Ki}} \right)}}$

The coupled ordinary differential equations that describe the reactionare:

$\frac{d\lbrack{cellobiose}\rbrack}{dt} = {- {rateBG}}$$\frac{d\lbrack{glucose}\rbrack}{dt} = {{+ 1.053}\mspace{14mu} {rateBG}}$

The concentration of cellobiose and glucose was defined in the units:g/L. The ordinary differential equations were solved numerically usingODE15s solver of MATLAB (the Mathworks, Inc., version 2012A), defaultoptions of the solver settings were used. The initial conditions forglucose cellobiose and enzyme concentrations were set according to theexperimental conditions. For each variant the following analysis wasapplied:

simulations were performed to predict the cellobiose to glucoseconversion for each of the 12 experimental conditions. The differencebetween model predictions and experimental data was minimized byestimating the values of kcat and K_(i) (K_(m) was set to 0.1 g/L.). Tothis end, nonlinear least square regression (MATLAB function Isqnonlin)was applied. As objective function, minimization of the differencebetween predicted and measured cellobiose and glucose concentrations wasused. Default settings of Isqnonlin were used except the option‘DiffMinChange’, which was set to 0.01.

Selection of mutants with improved kinetic properties was based on theestimated K_(i) and kcat values. The higher the value of Ki, the higherthe glucose tolerance (the lower the inhibition). The higher the valueof kcat, the faster is the variant: faster conversion of cellobiose intoglucose. The kinetic parameters Ki and kcat were calculated as unitlessparameters. The results are shown in Table 1.

The results demonstrate that variant polypeptide 3 is less sensitive toglucose inhibition than its parent polypeptide (the wild-typebeta-glucosidase), as the value for Ki is higher (3.2 vs 1.7). Theresults further demonstrate that variant polypeptide 1 is less sensitiveto glucose inhibition than its parent polypeptide (the wild-typebeta-glucosidase), as the value for Ki is higher (2.7 vs 1.7). Theresults demonstrate that variant polypeptide 2 is less sensitive toglucose inhibition than its parent polypeptide (the wild-typebeta-glucosidase), as the value for Ki is higher (2.6 vs 1.7). Theresults demonstrate that variant polypeptide 4 has improved kineticproperties (works faster) than its parent polypeptide (the wild-typebeta-glucosidase), as the value for kcat is higher (2.7 vs 1.0).

Example 3

Identification of Variant Polypeptides with a Higher Glucose Toleranceat 100 ml Scale

The hits selected from the MTP scale assay were subsequently furthercharacterized by monitoring cellobiose conversion at multiple timepoints at a larger scale. The beta-glucosidase having the amino acidsequence of SEQ ID NO: 2 was used as reference (parent/wild-typebeta-glucosidase). The assay was done as follows: the variantpolypeptides and the reference beta-glucosidase were incubated at aconcentration of 4.5 μg/ml in a 100 mM NaAc-buffer pH 4.5 with 30 mMcellobiose in a final volume of 100 ml for 2 hours at 62° C. undercontinuous stirring. Samples of 1 ml were taken at t=0 and after t=5min, t=10 min, t=15 min, t=30 min, t=45 min, t=60 min, t=90 min, t=120min and t=360 min and incubated for 5 minutes at 99° C. to stop thereaction. The glucose and cellobiose content of the samples wasdetermined using a High-Performance Liquid Chromatography System(Agilent 1100) equipped with a refection index detector (Agilent 1260Infinity). The separation of the sugars was achieved by using a 300×7.8mm Aminex HPX-87P (Bio rad) column; Pre-column: Micro guard Carbo-P (BioRad); mobile phase was HPLC grade water; flow rate of 0.6 ml/min and acolumn temperature of 85° C. The injection volume was 10 μl. The sampleswere diluted with HPLC grade water to a maximum of 2.5 g/l glucose andfiltered by using 0.2 μm filter (Afridisc LC25 mm syringe filter PVDFmembrane). The glucose and cellobiose were identified and quantifiedaccording to the retention time, which was compared to the externalglucose and cellobiose standards ranging from 0.2; 0.4; 1.0; 2.0 g/l.

The concentration cellobiose was plotted against time and the linearpart of this curve (the linear part of the cellobiose hydrolysis) wasused to calculate the V0 (the initial rate of cellobiose hydrolysis) inthe presence and absence of 20 g/l glucose. The ratio of these twocalculated rates (V0 in presence of glucose/V0 in absence of glucose)was calculated for the wild-type beta-glucosidase and the variantpolypeptides and is listed in Table 2. Variant polypeptides with ahigher ratio (V0 in presence of glucose/V0 in absence of glucose) thanthe wild-type beta-glucosidase have a higher tolerance against glucose(i.e. are less sensitive to glucose inhibition).

The results demonstrate that variant polypeptide 3 is less sensitive toglucose inhibition than its parent polypeptide (the wild-typebeta-glucosidase), as the ratio V0 in presence of glucose/V0 in absenceof glucose is higher (0.29 vs 0.18). The results further demonstratethat variant polypeptide 1 is less sensitive to glucose inhibition thanits parent polypeptide (the wild-type beta-glucosidase), as the ratio V0in presence of glucose/V0 in absence of glucose is higher (0.21 vs0.18). The results further demonstrate that variant polypeptide 2 isless sensitive to glucose inhibition than its parent polypeptide (thewild-type beta-glucosidase), as the ratio V0 in presence of glucose/V0in absence of glucose is higher (0.21 vs 0.18).

TABLE 1 Model prediction of Ki and kcat of variant polypeptides. ModelModel prediction prediction kcat Polypeptide Ki (unitless) (unitless)Wild-type beta-glucosidase (SEQ ID NO: 2) 1.7 1.0 Variant polypeptide 1(SEQ ID NO: 2 with 2.7 0.9 substitutions M90L + M335V + M485I) Variantpolypeptide 2 (SEQ ID NO: 2 with 2.6 1.1 substitution N103A) Variantpolypeptide 3 (SEQ ID NO: 2 with 3.2 0.8 substitution G142S) Variantpolypeptide 4 (SEQ ID NO: 2 with 1.1 2.7 substitution L606A)

TABLE 2 Ratio V0 in presence of glucose and V0 in absence of glucose ofvariant polypeptides. Ratio (V0 in presence of glucose/ V0 V0 V0 in(μmol/ml · (μmol/ml · absence min) min) of Polypeptide 0 g/l glucose 20g/l glucose glucose) Wild-type beta-glucosidase 0.52 0.09 0.18 (SEQ IDNO: 2) Variant polypeptide 1 0.72 0.15 0.21 (SEQ ID NO: 2 withsubstitutions M90L + M335V + M485I) Variant polypeptide 2 0.66 0.14 0.21(SEQ ID NO: 2 with substitution N103A) Variant polypeptide 3 0.55 0.160.29 (SEQ ID NO: 2 with substitution G142S)

1. A variant polypeptide comprising a substitution at a positioncorresponding to position 142 of the polypeptide of SEQ ID NO: 2,wherein the variant polypeptide has beta-glucosidase activity.
 2. Thevariant polypeptide according to claim 1, which is a variant of a parentpolypeptide which has beta-glucosidase activity and which comprises atleast 60% sequence identity to the polypeptide of SEQ ID NO:
 2. 3. Thevariant polypeptide according to claim 1, which comprises at least 60%sequence identity to the polypeptide of SEQ ID NO:
 2. 4. The variantpolypeptide according to claim 1, wherein the position corresponding toposition 142 is substituted to S.
 5. The variant polypeptide accordingto claim 1, which comprises substitution G142S.
 6. A polynucleotidewhich encodes the variant polypeptide according to claim
 1. 7. A nucleicacid construct or vector comprising the polynucleotide according toclaim
 6. 8. A host cell comprising the polynucleotide according to claim6 or a nucleic acid construct or a vector.
 9. The host cell according toclaim 8, wherein the cell is a fungal cell.
 10. A method of producingthe variant polypeptide according to claim 1, which method comprises: a)cultivating a host cell under conditions conducive to the production ofthe variant polypeptide, and b) optionally, recovering the variantpolypeptide.
 11. A composition comprising: (i) the variant polypeptideaccording to claim 1, and (ii) a cellulase and/or a hemicellulase and/ora pectinase.
 12. The composition according to claim 11, wherein thecellulase is selected from the group consisting of a lyticpolysaccharide monooxygenase, a cellobiohydrolase I, a cellobiohydrolaseII, an endo-beta-1,4-glucanase, a beta-glucosidase and abeta-(1,3)(1,4)-glucanase or any combination thereof and thehemicellulase is selected from the group consisting of an endoxylanase,a beta-xylosidase, an alpha-L-arabinofuranosidase, analpha-D-glucuronidase, an acetyl-xylan esterase, a feruloyl esterase, acoumaroyl esterase, an alpha-galactosidase, a beta-galactosidase, abeta-mannanase, a beta-mannosidase or any combination thereof.
 13. Thecomposition according to claim 11, wherein the composition is a wholefermentation broth.
 14. A method for the treatment of a substratecomprising cellulose and/or hemicellulose which method comprisescontacting the substrate with the variant polypeptide according to claim1 and/or a composition.
 15. A method of producing a fermentationproduct, which method comprises: a) treating a substrate using themethod according to claim 14, and fermenting the resulting material toproduce the fermentation product.